Biology Bulletin, Vol. 31, No. 1, 2004, pp. 75–79. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2004, pp. 86–91. Original Russian Text Copyright © 2004 by Dykman, Sumaroka, Staroverov, Zaitseva, Bogatyrev.
ANIMAL AND HUMAN PHYSIOLOGY
Immunogenic Properties of Colloidal Gold L. A. Dykman, M.V. Sumaroka, S. A. Staroverov, I. S. Zaitseva, and V. A. Bogatyrev Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entuziastov 13, Saratov, 410049 Russia e-mail:
[email protected] Received May 29, 2001
Abstract—We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund’s adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund’s adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.
INTRODUCTION The ability of colloidal gold (CG) to enhance immune response was previously used to produce antibodies against biotin (Dykman, 1996). In general, the problem of producing antibodies against partial antigens (haptens), including low molecular weight and poorly immunogenic compounds, remains among the current problems of modern immunology. Immunochemical tests are among the most convenient and sometimes the only available methods for monitoring antibiotics, hormones, vitamins, neurotransmitters, pesticides, and other compounds in the animal and human blood, meat and dairy products, and culture media. In addition, antibodies (Ab) against domains of biological macromolecules are an indispensable tool for studying their topography and structure. Application of Ab against low molecular weight compounds in immunotherapy also looks quite promising. Usually, Ab against haptens are produced after hapten binding to a polymer matrix, such as a protein (bovine serum albumin, ovalbumin, thyroglobulin, hemocyanin, etc.), polymer of the bacterial cell wall or flagellum (muramyl dipeptides and flagellin), or synthetic polyelectrolyte (poly-L-lysine, polyacrylic acid, and polyvinylpyrrolidone) (Kovalev and Polevaya, 1985; McEwen et al., 1992; Nesmeyanov et al., 1994; Petrov and Khaitov, 1998; Muller, 1999). However, this generates antibodies against both the hapten and the immunodeterminant sites of the carrier. Moreover, the immune response to weak antigens (Ag) is not always pronounced when such carriers are used. In addition, the subsequent isolation and screening of the generated
Ab are laborious and expensive, while their titer and affinity are often low. At first glance, the use of phage display is the most promising approach to the problem of producing Ab against haptens (McCafferty et al., 1990); however, this technique is virtually inapplicable in the modern vaccinology. “Complex Ag,” i.e. artificial macromolecular complexes including both essential epitopes and carriers (and/or adjuvants) are being developed. Such adjuvant carriers can deposit Ag at the site of injection, improve Ag presentation to immunocompetent cells, and induce the synthesis of the required cytokines. Corpuscular carriers are particularly promising among such adjuvants: polymeric nanoparticles (e.g., polymethylmethacrylates) (Kreuter, 1995), liposomes, microcapsules (Fukasawa, 1998), and even fullerenes (Andreev et al., 2000). In addition, several authors have reported successful production of Ab against certain haptens (amino acids, neuromediators, and vitamins) mediated by application of CG as a carrier (Shiosaka et al., 1986; Tomii et al., 1991; Pow and Crook, 1993; Dykman et al., 1996; Chen et al., 2000), while CG is a traditional label for conjugation with a recognition probe in immunochemistry (Colloidal Gold, 1989; Dykman and Bogatyrev, 1997). According to these data, such Ab had a high affinity to the studied Ag and a higher titer (“extremely high” according to Pow and Crook (1993)) as compared to the Ab produced by routine methods. What a priori advantages are offered by CG particles as an Ag carrier during immunization, and why we decided on them? It is common knowledge that gold hydrosols are typical lyophobic colloids stable only at
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Table 1. Description of the antigens used for immunization (Ag + CG + CFA scheme) Antigen Bacteriorhodopsin BSA Actin Chloramphenicol Gentamicin Ivermectin Tilmicosin Peptide 1 Peptide 2 Peptide 3 c-myc peptide
pH 10 6 6 10 10 10 11 10 6 5.5 6
Gold num- Ag amount per ber, µg/ml injection, µg 5 10 10 – – 15 8 – – 10 5
0.25 0.5 0.5 125 125 0.75 0.4 25 25 0.5 0.3
low ionic strength. Electrostatic and “hydrophobic” interactions provide for gold binding by compounds of various nature. It is important to emphasize here that relatively small quantities of the compounds have a stabilizing effect (against salt aggregation) on the system: 5–10 µg/ml sol for the standard concentration of gold (~5 µg/ml) and traditional particle size (15–30 nm). Such weak (noncovalent) interactions largely preserve the native structure of the biomolecules, and hence, make possible their presentation to immunocompetent cells in a virtually unchanged form. Finally, CG per se is neither allergenic nor toxic. The goal of this work was to evaluate the efficiency of CG as a tool for Ab production in vivo for the maximum possible number of Ag of various nature in order to standardize the technique and to clarify adjuvant activity of CG. MATERIALS AND METHODS Antigens used for immunization. The following high molecular weight proteins were used as complete Ag: bacteriorhodopsin isolated from Halobacterium halobium membrane (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences), chicken smooth muscle actin (Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences), and bovine serum albumin (BSA; Sigma, United States). A number of antibiotics of different chemical composition, including chloramphenicol, gentamicin, ivermectin, tilmicosin (NITAFARM, Russia), were used as haptens. In addition, we used three synthetic bacteriorhodopsin peptides and one synthetic peptide of human c-myc proto-oncogene (hereinafter, c-myc peptide) (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences).
Preparation of Ag–CG conjugates. CG with the mean particle diameter of 20 nm was obtained according to Frens (1973) by reduction of tetrachloroauric acid (Aldrich, United States) with sodium citrate (Fluka, Switzerland). The buffer pH was selected individually for each Ag (Table 1). The conjugation was carried out by simple mixing (without special coupling agents) of gold sol and Ag in the concentration determined using the gold number (Colloidal Gold, 1989; Dykman and Bogatyrev, 1997). The conjugates stability was assessed by the absence of aggregation (color change from red to blue or gray) after adding 10% aquatic solution of NaCl to a final concentration of 0.5%. Protocol of animal immunization. Balb/C mice at the age of four months, adult female rats, and Chinchilla rabbits were used for immunization. Immediately prior to immunization, an Ag–CG conjugate was mixed with complete Freund’s adjuvant (CFA) (Sigma, United States) at a 1 : 1 ratio. The resulting emulsion was injected intraperitoneally into mice and rats or subcutaneously into rabbits in the dose of 100 µl. The injections were repeated four times with the interval of two weeks. Ten days after the last injection, the blood was drawn to collect the serum. The immunoglobulin fraction was salted out with ammonium sulfate as described (Antitela, 1991). Alternatively, the animals were immunized with a CG-conjugated Ag, CFA–Ag mixture, or Ag suspension in saline. Testing the produced Ab. The serum titer was determined by hemagglutination reaction. Conjugation of proteins to the erythrocytes was carried out using glutaraldehyde (Immunologicheskie metody, 1987). The resulting Ab were tested by dot assay (Dykman and Bogatyrev, 1997) using CG (or protein A) -conjugated antimouse Ab as a secondary marker (Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences). Dot assay was carried out on nitrocellulose membranes with a pore size of 1.5 µm (Synpor, Czech Republic) or polyvinylidene fluoride membranes with a pore size of 0.22 µm (Millipore, United States). A test sample (1 µl) was applied onto a membrane strip in the center of a marked square of side 5 mm. For better adsorbate binding, the membranes were incubated in a dry air chamber at 60°C for 15 min. Nonspecific adsorption centers were blocked by incubation of the membranes with the samples for 30 min at room temperature in a blocking buffer containing 2% defatted dry milk, 150 mM NaCl, and 20 mM Tris–HCl, pH 8.2. The Ag were revealed by incubation of thus treated membrane strips in solutions of the corresponding Ab (the typical concentration was 10 µg/ml) in Parafilm envelopes for 1 h at room temperature. After washing in the buffer, the strips were incubated with CG-labeled secondary Ab diluted to optical density λ520 = 0.5. The reaction was manifested as red spots at the site of specific interaction between Ag and homologous Ab (revealed by CG-labeled anti-immunoglobulins) 3–5 min after addiBIOLOGY BULLETIN
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tion of the marker; it gradually developed for 1 h. Then the strips were removed and washed with water. After that, they could be stored for any time without color fading. Normal (nonimmune) mouse serum and the serum of control mice “immunized” with a CG or CG + CFA solution using the protocol of the experiment were used as the control. Evaluation of natural resistance factors. Ten days after the second, third, and fourth immunizations, the blood of experimental animals was collected to evaluate the factors of natural cellular and humoral resistance. The assays for functional and bactericidal activity of the phagocytes and for the serum contents of lysozyme and complement were carried out as described elsewhere (Paster et al., 1989). RESULTS The most amazing and surprising result was a high Ab titer for all used Ag, which varied from 1 : 6400 to 1 : 51 200. Most problems were related to the attachment of haptens to the gold particles. In particular, conjugation of virtually water-insoluble ivermectin and tilmicosin to CG was carried out in the presence of polypropylene glycol. In addition, not all haptens had stabilizing effect on gold sol, and consequently, the gold number could not be determined. In such cases, we mixed 0.1–0.5% haptens and CG at a 1 : 1 ratio. Thus, the quantities of Ag used for each injection varied from 0.25 to 125 µg (Table 1). Nevertheless, these quantities were lower than those used for hapten–protein conjugates or complete Ag. Table 2 demonstrates an increase in the serum titer in the course of immunization of rats with BSE according to the above-mentioned schemes. Note that the maximum titer after the immunizations was specific for the serum obtained by injection of BSA–CG conjugate without CFA. Figures 1–4 show changes in the nonspecific factors of cellular and humoral resistance during immunization of rats with BSA according to various schemes. A B C
Serum activity, % 150 100
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Table 2. Time-related changes in the serum titers in the course of rat immunization with BSA according to different schemes 2nd immuni- 3rd immuni- 4th immunization zation zation
Sample BSA + CG BSA + CFA BSA
1 : 128 1 : 128 1 : 128
1 : 2560 1 : 2560 1 : 1280
1 : 6400 1 : 3200 1 : 2560
Figures 5 and 6 present immunodot assay of the c-myc peptide and actin using Ab produced by immunization with native Ag, native Ag with CFA, Ag–CG conjugate, Ag–CG conjugate with CFA, and Ag–BSA conjugate (for the peptide). It can be seen that the minimum Ag quantity (both hapten and complete Ag) detectable by the dot assay using the sera obtained with CG is no less than that for the sera obtained by routine methods. No positive response was obtained for mouse immunization with the c-myc peptide either with or without CFA. Note the positive response for the Ag–CG conjugates even in the absence of CFA. This fact suggests that the gold particles may have an immunomodulating (adjuvant !?) activity. DISCUSSION Thus, we demonstrated the possibility of in vivo generation of At against complete Ag and haptens of various nature using CG particles as a carrier. In addition, Ab production in response to CG conjugates with BSE, actin, and c-myc peptide in the absence of CFA can indicate adjuvant properties of CG proper, which is clearly promising for developing a new generation of vaccines. CG application as an Ag carrier proved to induce phagocytic activity of the lymphoid cells, which can be responsible for its immunomodulating activity. Indeed, we used various compounds as Ag attached to CG; these compounds differed in their molecular weight (42 000 for actin and ~700–1000 for antibiotics Complement titer, rel. units/ml
100
A B C
80 60 40
50 0
20 0 2nd
3rd Immunization
4th
Fig. 1. Changes in serum bactericidal activity in the course of rat immunization with BSA according to different schemes: (A) BSA + CG, (B) BSA + CFA, (C) BSA (for Figs. 1–4). BIOLOGY BULLETIN
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3rd Immunization
4th
Fig. 2. Changes in the serum content of complement in the course of rat immunization with BSA.
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A B C
15 10 5 0
Phagocytic activity, % 100
A B C
80 60 40
2nd
3rd Immunization
20
4th
0
1-fl
2nd Immunization
3rd
Fig. 4. Changes in the serum phagocytic activity in the course of rat immunization with BSA.
Fig. 3. Changes in the serum content of lysozyme in the course of rat immunization with BSA.
(a) (b) (c) 1
3
2
Fig. 5. Immunodot assay for (a) c-myc peptide, (b) c-myc peptide–BSA conjugate, and (c) native BSA with mouse antibodies obtained after immunization with (1) c-myc peptide–CG conjugate, (2) c-myc peptide–CG–CFA conjugate, and (3) c-myc peptide– BSA–CFA conjugate; CG-labeled antimouse antibodies were used as a secondary marker.
and peptides), chemical nature (polypeptides and polyheterocyclic compounds), and physicochemical properties. Recall that attachment of macromolecules to solid surfaces is mediated by Van der Waals, Coulomb, and “hydrophobic” forces. At the same time, one should distinguish between the attachment of macromolecules to gold particles and the stabilizing effect (protecting from salt aggregation). In our case, it was important to provide for the adsorption of macromolecules on CG particles. When macromolecules had no protective effect on gold sol (chloramphenicol, gentamicin, and bacteriorhodopsin peptides 1 and 2), the interaction was estimated from CG aggregation that occurred at pH below the values indicated in the table even without salt addition. Moreover, the markers obtained by conjugation with Ag specifically reacted with the homologous antisera. Cross reactions were observed only in the experiments with ivermectin and tilmicosin. Nevertheless, we believe that these data confirm firm attachment of macromolecules with molecular weight below 1000 to the gold particle surface, which is sufficient for their presentation to immunocompetent cells. This fact can be verified by the methods of oscillation spectroscopy (Dou et al., 1999). We realize that adsorption of macromolecules is a dynamic process, and the substances with a higher affinity to gold, such as serum proteins, can displace them from the gold particle surface; however, the adsor-
bate nature does not seem to have considerable effect on CG adjuvant activity. In the case of actin, a good stabilizer for CG and, at the same time, a poorly immunogenic Ag (due to its evolutionary conservatism), our experiments demonstrated an increased immune response for the actin–CG conjugate (Fig. 6). The problem of the mechanisms underlying such activities of gold particles remains open. The arguments of Pow and Crook (1993) concerning preferential macrophage response to corpuscular Ag are clearly valid but do not resolve the problem of further mechanisms of Ag presentation to T helpers. The theory of multivalent Ag mentioned in some publications gives no clue to this problem as well.
1 2 3 4 Fig. 6. Immunodot assay for actin with mouse antibodies obtained after immunization with (1) native actin, (2) actin + CFA, (3) actin–CG conjugate, and (4) actin–CG–CFA conjugate; CG-labeled antimouse antibodies were used as a secondary marker. BIOLOGY BULLETIN
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It is quite probable that CG effect as a chemotherapeutic drug for rheumatoid arthritis is also explained by its capacity to enhance nonspecific immune response (Abraham and Himmel, 1997). The mechanisms underlying immunomodulating activity of CG particles will be addressed in our future studies. ACKNOWLEDGMENTS We are grateful to Prof. V.A. Nesmeyanov (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences), Dr. O.I. Sokolov (Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences), S.V. Semenov, and D.A. Zhemerichkin (NITA-FARM, Russia) for providing various antigens. We also thank D.N. Tychinina and Yu.V. Gogoleva (Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences) for their help in manuscript preparation. This work was partially supported by the Russian Foundation for Basic Research, project no. 01-04-48736, and CRDF grant no. REC-006. REFERENCES Abraham, G.E. and Himmel, P.B., Management of Rheumatoid Arthritis: Rationale for the Use of Colloidal Metallic Gold, J. Nutr. Med., 1997, vol. 7, pp. 295–305. Andreev, S.M., Babakhin, A.A., Petrukhina, A.O., Romanova, V.S., Parnes, Z.N., and Petrov, R.V., Immunogenic and Allergenic Properties of Fulleren Conjugates with Amino Acids and Proteins, Dokl. Ross. Akad. Nauk, 2000, vol. 370, pp. 261–264. Antibodies—A Practical Approach, Catty, D., Ed., Washington: IRL, 1989. Translated under the title Antitela, Moscow: Mir, 1991. Chen, J., Zou, F., Wang, N., Xie, S., and Zhang, X., Production and Application of LPA Polyclonal Antibody, Bioorg. Med. Chem. Lett., 2000, vol. 10, pp. 1691–1693. Colloidal Gold: Principles, Methods and Applications, Hayat, M.A., Ed., San Diego: Academic, 1989, vol. 1. Dou, X., Jung, Y.M., Yamamoto, H., Doi, S., and Ozaki, Y., Near-Infrared Excited Surface-Enhanced Raman Scattering of Biological Molecules on Gold Colloid I: Effects of pH of the Solutions of Amino Acids and of Their Polymerization, Appl. Spectrosc., 1999, vol. 53, pp. 133–138. Dykman, L.A. and Bogatyrev, V.A., Colloidal Gold in SolidPhase Assays: A Review, Biochemistry (Moscow), 1997, vol. 62, pp. 411–418.
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