The avidin-biotin complex in immunology

The avidin-biotin complex in immunology

39 Immunology Today, vol. 5, No. 2, 1984 cytes and antibody-coated target cells 14. Such release would be most effective if the respiratory burst was...

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Immunology Today, vol. 5, No. 2, 1984 cytes and antibody-coated target cells 14. Such release would be most effective if the respiratory burst was triggered locally by cell or parasite contact. This brings home the value of antibodies and/or complement in ensuring the maximum contact between effector and target cell. As a final illustration of the benefits of close proximity we might cite the malaria parasite itself. In an electronmicroscope study of the bone marrow during Plasmodium berghei infection in mice, Weiss 15 has found that infected reticulocytes positioned themselves on the vascular sinus endothelium in close apposition to freshly developing reticulocytes so that individual merozoites ('favoured' in

Weiss's phrase) could reinvade without exposure to antibody and other anti-parasite factors in the plasma. The message seems to be that when thinking of cell- cell interactions, it is probably better to visualize a crowded cocktail bar than a half-empty swimming pool. j. H. L. PLAYFAIR H. M. DOCKRELL R. LELCHUK

Departmentof Immunology, The MiddlesexHospitalMedicalSchool, London W1P 9PG, UK.

References 1 Smith, K. A. (1980) ImmunoL Rev. 51,337 2 Wagner, H., Hardt, C., Heeg, K. et al. (1980)Imrnunol. Rev. 51, 215 3 Hardt, C., Rollinghoff, M., Pfizenmaier, K. et aL (1981)J. Exp. Med. 154, 262 4 Malkousky, M., Asherson, G. L., Stockinger, B. and Watkins, M. C. (1982) Nature (London) 300, 652 5 Kramer, M. and Koszinowski, U. (1982)o( ImmunoL 128, 784 6 Klassen, L. W., Ahmann, E. B. and Ballas, E. K. (1982) Fed. Proc. Fed. Am. Soc. Exp. BioL Med. (abstract) 41,563 7 Eautam, S. C., Hilfiker, M. L. and Battisto, J. R. (1983),]. Immunol. 130, 533 8 Lelchuk, R. and Playfair, J. H. L. (1980) Clin. Exp. Immunol. 42, 428

9 Tada, T. and Okumura, K. (1980) Adv. ImmunoL 28, 1 10 Nathan, C. F. (1980) in Mononudear Phagoo~tes - Functional Aspects (Van Furth, R., ed.), p. 1165, Martinus Nijhaff, The Hague 11 Nathan, (3. F. (1983) Trop. Med. Hyg. 77, 620-630 12 Dockrell, H. M. and Playfair, J. H. L. (1983) Infect. Immun. 39, 456-459 13 Silverstein, S. C., Michl, J., Nathen, C. F. and Horwitz, M. A. (1980) in Basic and Clinical Aspects of Granulomatous Diseases (Boros, D. L. and Yoshida, T., eds), p. 70, Elsevier, North Holland 14 Seim, S. and Espevik, T. (1983) ~L Reticuloendothelial Soc. 33, 417-428 15 Weiss, L. (1983)J. Parasitol. 69, 307-318

The avidin-biotin complex in immunology Meir Wilchek* and Edward A. Bayer The tenacious interaction between the vitamin biotin and the glycoprotein avidin has beenapplied in many immunological systems for the isolation, localization and visualization of various antigens as well as drug delivery, lymphocyte stimulation and immunoassays. These are discussed here by Meir Wilchek and Edward Bayer, who emphasize that with the increasedr(inement of affinity systems, the avidin-biotin complex will undoubtedly enable improved detectability and determination of minute amounts of antigens. The avidin-biotin complex has no known natural role in immunology. Biotin is a vitamin, and as such (as other vitamins) may have indirect importance to the immune system. O n the other hand, the glycoprotein avidin may indeed play an important antimicrobial role in the immune system during tissue injury and embryonic development I, although the precise nature of such a role has yet to be defined. The major link of the avidin-biotin complex to immunology is but an artificial one of scientific application, and is an outgrowth of the remarkable affinity of the glycoprotein for the vitamin 2. The four binding sites together with the high affinity (KD = 10 - 15M - i) ofavidin for biotin serve as an aid in amplifying the sensitivity of immunoassays which have become so widespread as a general diagnostic tool in all fields of biology. Based on the strong interaction, the avidin-biotin complex has also been used as a tool for drug delivery via tumor-specific antibodies, for elucidating the mechanism of non- specific polyclonal activation of lymphocytes, and in the isolation Department of Biophysics, The Weizmann Institute of Science, 76100 Rehovot, Israel. *Fogarty Scholar-in-Residence, NIH, Bethesda, MD, USA.

and localization of various surface constituents on the lymphocyte and other cells of the immune system. A simplified schematic diagram showing the general application of the avidin-biotin complex in immunological systems is presented on the centre pages of this issue. The varied uses of the avidin-biotin complex in other fields of biology have recently been reviewed by us 3'~.This approach was made possible by the availability of various avidin-conjugated probes as well as many group-specific biotin derivatives which can be covalently coupled to antigens, antibodies, lectins, hormones, nucleic acids, cells and subcellular organelles. The preparation of these derivatives and probes has been described previously and will not be discussed here. Localization The first reported application of the avidin-biotin complex in an immunological system was its use in an immunohistochemical study, whereby biotin-labelled antibody in conjunction with ferritin-avidin conjugates were employed for the localization of erythrocyte surface antigens 5. It immediately became clear that this approach holds distinct advantages over other systems (even direct coupling), since the preparation of purified immuno© 1984,ElsevierSciencePublist~l~B.V, Amsterd~ 0167- 4919/84/$02.00

40 globulins was unnecessary, i.e. the biotinylation procedure could be performed even on whole antiserum. In other systems, the appropriate antibody would first be purified in order to facilitate subsequent protein-protein conjugation. Ferritin-avidin conjgates were also used in conjunction with biotinylated antibodies both to study cell structures involved in the synthesis ofopsin in frog photoreceptor cells 6 as well as for the localization of cell-surface antigens in bacterial cells 7. A similar approach was used later by Warnke and Levy 8. Using monoclonal antibodies, the authors were able to detect the presence of Ialike antigens on three types of B-cell lymphomas, and tested the presence of two T-cell antigens on three types of T-cell lymphomas. This was accomplished by binding the monoclonal antibodies to cells followed by biotinylated goat anti-mouse antibody. Upon further incubation with avidin-conjugated horseradish peroxidase, cells bearing the specific surface antigen could be stained subsequently with diaminobenzidine. All of the B cells stained heavily for Ia antigen, whereas the T cells were shown to be heterogeneous for the expression of the two T-cell antigens. The authors claim that this approach may prove useful in the immunological characterization of human lymphomas. The technique using the avidin-biotin complex proved more sensitive and more specific than conventional enzyme-labelled antibody techniques. The use of the avidin-biotin-peroxidase complex has also been adapted by Hsu and colleagues for different histochemical localizations of surface receptors using hormones, antibodies or other binding proteins 9'1°. Monoclonal antibodies of defined specificity (for example, Lyt-1, mouse T-cell differentiation antigen), which have been biotinylated directly, have been produced commercially (Becton Dickinson FACS Systems, Sunnyvale, CA). The biotinylated monoclonals are appropriate for use in localization as well as in cell-separation studies, using fluorescent avidin (fluorescein or rhodamine) in conjunction with fluorescence-activated cell sorter (FACS) analysis. In order to further enhance the fluorescence of localized antigens, a sandwich-type assay has been used, i.e. successive treatments with biotinylated antibody, fluorescent avidin and fluorescent anti-avidin antibodies. Using this approach, Berman and Basch H achieved a fivefold enhancement of staining over that achieved by the conventional technique of immunofluorescence. The brightness was more than twice that obtained by the usual interaction of the biotinylated conjugate with fluorescent avidin. In a somewhat similar approach 12, purified human complement C3 was biotinylated and shown to retain over 90% of its specific hemolytic activity. When bound to sheep erythrocytes, the biotinylated complement maintained the ability to adhere to human C3B receptors. This binding could be inhibited by avidin. Fluorescent avidin reacted with cell-bound biotinyl C3B and could be used to quantify the cell-bound C3 of individual cells by flow cytometry. Ferritin-conjugated avidin was also used in this system as a marker to characterize the ultrastructural distribution of the C3B receptors. This approach should eventually help in understanding the reactivity of complement with heterogeneous cell populations, for example tumor cells. Of course, this system

Immunology Today, vol. 5, No. 2, 1984

could also be used for the isolation of the receptor using an avidin column 53. Using the same approach, the antibodyinduced fixation of complement to the cell walls of encapsulated pneumococci has also been investigated via the avidin-biotin complex 14.

Immunoassay The first prototype study which demonstrated that the avidin-biotin system could be used in immunoassays was a study15 in which it was shown that biotinylatcd bacteriophage can be inactivated by avidin. The inactivation could be reversed by biotin and derivatives in solution, similar to the interaction of phages using antibodies and antigens 16. The realization that the avidin-biotin complex could be effectively used for imrnunoassays was first demonstrated by the work of Avrameas and co-workers 57 in which they suggested several related methods for its use in increasing the sensitivity and specificity of solid-phase immunoassays (especially enzyme-linked immunoassays). Two different approaches were proposed. In both cases an antibody is immobilized onto a carrier to which an antigen is added. In one method, a biotinylated preparation of the same antibody is added to the immobilized antigen. Subsequent incubation with native avidin is followed by biotinyl enzyme (for example, alkaline phosphatase, peroxidase, galactosidase or glucose oxidase). Here avidin acts as a bridge between the biotinylated antibody and the biotinylated enzyme. In the second approach, treatment with the biotinylated antibody was followed by introduction of avidin covalently conjugated to the appropriate enzyme. Both methods gave results comparable to those of other immunoassay methods, but the advantage of the first approach is that the preparation of protein-protein conjugates is not required. A related study employing the avidin-biotin complex in combination with an erythroimmunoassay has also been described 58. In addition, the system has been modified for competitive enzyme immunoassay59. It will be very interesting to see whether sensitivity would be further increased if two different monoclonal antibody preparations (each specific for a different nonoverlapping determinant on the same antigen) would be used. In this approach, one species of monoclonal antibody would be immobilized to the carrier and the antigen would then be introduced to the experimental system. The second species of monoclonal antibody would then be derivatized with biotin and added to the system and analysed or probed via subsequent incubation with an appropriate avidin conjugate. Undoubtedly, this notion is currently being pursued. In our laboratory, we initiated solid-phase radioimmunoassay using the avidin-biotin complex 2°. Unfortunately, since tyrosines are important to the biological activity of avidin 2, iodination of avidin is accompanied by a concomitant loss of biotin-binding activity, and consequently only limited amounts of radioiodination significantly reduces the sensitivity and specificity of the assay. This limitation can be overcome, however, either by the use 25of the Bolton-Hunter reagent which binds to tysine groups of avidin or by iodination of polytyrosine-conjugated avidin thereby achieving high specific activity (unpublished observations). It would therefore be interesting to repeat the radioimmunoassay

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study using highly iodinated preparati6ns of avidin and to determine the comparative sensitivity of this assay with the enzyme immunoassay. Recently, the avidin-biotin system has been adapted for the clinical detection of herpes simplex infections 22. H u m a n tissue cultures were infected with suspected herpesvirus and stained successively with biotin-bound herpes-specific antibodies followed by avidin-fluorescein conjugates. As a result of the staining process, any herpesvirus in the culture becomes fluorescent and can be observed microscopically. Using this strategy, the quality of fluorescence was far superior to that of conventional fluorescent-antibody techniques and the method may of course be applied to other viruses. In summary, both in localization studies and in immunoassay studies, the avidin-biotin complex has several advantages: (a) only one protein-protein (avidin marker) conjugate needs be prepared for all affinity systems; (b) selective purification of individual antibodies is unnecessary (whole antiserum may be used); (c) biotin can be attached to antibody under mild conditions; (d) the size, physical characteristics and biological activity ofbiotinylated antibodies are only nominally affected; (e) the avidin-biotin complex is of exceptionally high affinity and stability; (f) the introduction of biotin groups into antibodies followed by the derivatized or conjugated avidin probe leads to amplified detectability of the antigen; (g) avidin, biotin, and various conjugates are readily available from a variety of commercial sources*.

Immunotherapy In recent years, a novel approach for the potential treatment of cancer was introduced in which antibodies were tested as carriers for cytotoxic drugs or for polypetide toxins 23. Such an approach has advantages, since cytotoxic drugs are usually as toxic to normal cells as to cancer cells, and the inherent toxicity limits the effective use of chemotherapy in treatment of neoplastic diseases. A delicate balance of the drug must therefore be supplied in an attempt to selectively kill the cancer cells. The approach using antibodies specific to tumor-associated antigens should thus increase the local concentration of the drug on tumor cells thereby effecting site-directed killing of the cancer cells. This approach, however, suffers several drawbacks. For example, the amount of drug which can be attached to the antibody without losing activity or precipitating the conjugate is limited. In order to circumvent this problem, the avidin-biotin complex was used to enhance the amount of drug that can be effectively delivered to the cell surface: for a given amount of biotinylated anti-cancer antibody molecule, several molecules of toxin or drug conjugated to avidin can be introduced to the system. The amount can be even further increased if biotinylated drug or toxin is added over and above the original system. Such an approach was recently taken by Philpott et al. 24 using RBL1 basophilic leukemia *Among the companies which provide avidin and biotin products are the following: Becton Dickinson FACS Systems, Sunnyvale, CA; S. C. Belovo, Bastogne, Belgium; Enzo Biochem Research Products, New York, NY; E. Y. Laboratories Inc., San Mateo, CA; Miles Laboratories Ltd, Elkhart, IN; Pierce Chemical Co., Rockford, IL; Reactifs IBF, Villeneuve-la-Garenne, France; Sigma Chemical Co., St Louis, MO; and Vector Laboratories Inc., Burlingame, CA.

41 cells. These cells possess a surface receptor specific for IgE immunoglobulin. To this immunogiobulin biotin was bound covalently, and the cells were treated with the biotinylated IgE. Such treatment was followed by successive incubations with avidin-glucose oxidase con= jugates, lactoperoxidase, and potassium iodide. The system worked as follows: after binding biotinylated IgE and the avidin-enzyme conjugate to the cells, hydrogen peroxide is produced in the presence of glucose. Iodide is oxidized to iodine and the cells are heavily iodinated by the action of lactoperoxidase. In this system, more than 90% of cells possessing the receptor were killed. Of course, the efficiency of killing can be further increased using radioactive iodide. The authors claim that in using this system they have elevated the cytotoxic potential by several factors: the ratio of biotin to immunogiobulin in the conjugate was 10/1, whereas in previous works the efficiency of conjugating IgE to the enzyme was only 10-20 % even in the best preparations. One can immediately see the enhancement factor if it is assumed that each biotin binds one avidin-enzyme conjugate. As mentioned earlier, the conjugation of biotin to IgE causes little reduction in the specificity or binding capacity of the antibody to the cell surface. Additionally, the biotin-coupled IgE did not greatly affect the distribution properties of IgE when injected intracardially. This system was shown to be very efficient in vitro, whereas in vivo one must take into consideration, as the authors claim, that normally occurring mast cells are found in various organ systems throughout the body. These naturally occurring mast cells also possess IgE receptors and consequently these tissues, for example peritoneum, gastrointestinal tract and skin, may be adversely affected by this treatment. In a similar approach, Urdal and Hakomori 25 used antibodies to tumor-associated glycolipids to deliver a toxic drug, neocarzinostatin, to tumor cell surfaces. In order to increase the amount of drug, avidin was again used to bridge between the biotinylated drug and the biotinylated antibody bound to the surface antigen. An even further increase in drug delivery was attempted by incorporating either biotin or avidin onto drug-encapsulated lipsomes. Although the targeting of the liposomes to the cell surface was very successful, subsequent killing efficiency was limited, apparently due to the subsequent lack of fusion between the liposome and the cell membrane26 In our laboratory we have also attempted to use biotinylated antibodies or biotinylated monoclonal antibodies to tumor:associated antigens, followed by avidinricin conjugates or avidin-daunomycin. The problem with this sequential approach is that in all these studies the effectivity depends upon the ability of the avidin-drug to find the biotin-antibody bound to the cancer cell in vivo, since if supplied together, avidin may inhibit the bridging of the biotin-conjugated antibody to the cell surface. Further efforts in this regard will therefore be necessary before the system will be of general applicability. It is also not clear what influence endogenous biotin 27might have either as an inhibitor of the targeting of the avidin to the cancer cell, or as an alternative target. Lymphocyte activation It is well established that in the presence of various

42 agents (mitogens) the resting lymphocyte is activated to undergo blastogenesis. It is generally considered that the activation signal is confined to glycoconjugates of the cell membrane, since either lectins or periodate oxidation, both of which interact with specific carbohydrate moieties on the cell surface, cause stimulation of the cells 28. The avidin-biotin complex has been instrumental in establishing the unequivocable involvement of surface carbohydrate groups in the activation process 29, This was achieved by trying to determine whether specific mitogenic sites really do exist or whether a simple perturbation of the membrane is sufficient to induce blastogenesis. In order to answer this question we tried to ascertain the respective rote of the protein portion versus that of the carbohydrate portion of the membrane-based glyeoconjugates by modifying each with group-specific reagents containing covalently attached biotin. It was assumed that if perturbation of the membrane was sufficient, avidin - which would cross-link the biotin sites - would in every case stimulate the lymphocyte. Surprisingly, it was found that the introduction of biotin to functional groups of various amino acids (for example lysine, cysteine and tyrosine) of membrane proteins failed to stimulate cells in the presence of avidin, even though the cells were still viable and amenable to conventional mitogenic stimulation. This was not due to the absence of modified membrane-based proteins, since avidin caused agglutination and fluorescent-avidin or ferritin-conjugated avidin labelled the biotinylated surface extensively. On the other hand, when sugar moieties were modified by periodate oxidation to form aldehyde groups followed by treatment with biotin-hydrazide, a high stimulation was achieved upon introduction of avidin; thereby providing concrete evidence that the carbohydrate groups of glycoconjugates are essential intermediates in lymphocyte stimulation. Another question which is frequently asked is whether cross-linking is important in the stimulation. Conflicting reports concerning this point have been published. In this case the avidin-biotin complex alone could not provide an answer since monovalent avidin could not be prepared to decide this dispute. The avidin-biotin complex ultimately proved useful in this regar& °, however, by using another biotin-binding protein, i.e. monovalent Fab derived from anti-biotin antibodies 3~. It was shown that (in contrast to the dimer) the Fab monomer is non-stimulatory, pointing to the importance of multivalency towards lymphocyte activation. To further prove this point the cells were initially stimulated by another hapten-hydrazide derivative [dinitrophenylated hydrazide (DNP-hydrazide)]. Again it was possible to show that the anti-DNP Fab monomer was non-stimulatory. However, upon biotinylating the Fab monomer, followed by treatment with avidin, stimulation was restored, thus proving that cross-linking is essential to the stimulatory process. Since the carbohydrate portion of the lymphocyte membrane is composed of both glycoproteins and glycolipids, the question remaining was to what extent is each of the two glycoconjugates involved in the stimulation of lymphocyte cells 32. Using lectins or periodate, this is difficult to demonstrate since a certain amount of structural overlap is inherent in the two species, especially in the terminal oligosaccharide sequences. Sequential treatment of

Immunology Today, vol. 5, No. 2, 1984

cells with periodate and biotin-hydrazide would modify both species of glycoconjugate. Consequently, it would be impossible to ascertain the exact role of each in the process. In order to evaluate the role of gangliosides, a novel approach was taken 3~. A mixture of bovine brain gangliosides was treated with periodate and biotin-hydrazide in vitro. The reactions were essentially the same as those applied to the whole cell. These biotin-modified gangliosides were incorporated into the lymphocyte membrane and stimulation was readily obtained upon addition of avidin. Agglutination, patching, and capping of cells bearing exogenously incorporated biotinyl gangliosides were demonstrated to accompany the observed stimulation. It was possible to show that the gangliosides were incorporated in the correct orientation by using ferritinavidin conjugates ~4'35,and to determine the topographical localization of the glycolipids relative to that of the glycoproteins in the membrane 36. These studies point to the possibility that the glycolipids are actively involved (either alone or in concert with glycoproteins) in the stimulation of lymphocytes. F u t u r e p r o s p e c t s a n d c o n c l u d i n g remarks

It is clear that the avidin-biotin complex will continue to be used in all systems in which affinity reactions are involved. Sophisticated approaches will eventually be taken to increase the sensitivity of both homogeneous and heterogeneous immunoassays. It is also clear that this system is finding general use as an aid to enhance a given experimental response, to unify the various approaches, or as a secondary affinity probe for localization purposes in fluorescent and electron-microscope studies 13. It will also be used as an aid for the separation, elimination or retrieval of specific subpopulations of cells ~7'38. Such an approach has already been taken in our group by binding biotin to lectins or antibodies and enhancing the agglutination and precipitation of cells containing the appropriate receptors. Interestingly, due to the attraction of the system, researchers are attempting to apply the avidin-biotin complex to solve a given problem, but are surprised to discover that the system is not ubiquitously suitable. In this context, it should be kept in mind that avidin is a basic glycoprotein which is also recognized by various lectins and subject to electrostatic interaction with acidic macromolecules, thereby leading in some systems to nonspecific interactions. Since these researchers have already gone to the trouble of adapting their system to biotin, antibiotin antibodies have then been prepared and substituted for avidin, even though any other hapten would have been suitable in its place. Such a study was recently described in developing a new method for gene mapping, using biotinylated uridine and anti-biotin antibodies 39. The availability of two types of biotin-binding proteins - one naturally occurring (i. e. avidin or streptavidin), and the other artificially induced (i.e. anti-biotin antibodies) may open new avenues of application to the system. The versatility of this system is even further amplified upon preparing anti-avidin antibodies 4°, since now we have a protein which can both bind a small ligand and be recognized itself by another binding protein. In fact, a very exciting recent development involves the


Immunology Today, vol. 5, No. 2, 1984

use of the avidin-biotin complex as ~ tool for studying molecular mechanisms by which antigens are processed and presente d to T lymphocytes 41. In this study, avidin was used as an-a~tigen and, after being processed by antigen-presenting cells, was still able to recognize the biotin molecule4L The processed avidin was a thousandfold more efficient than native avidin as an immunogen for primed T lymphecyteso This system may eventually be useful in attempts to isolate the T-cell receptor. The virtually unlimited possibilities inherent in the avidin-biotin system warrants further development and application in all fields of biology.

References 1 2 3 4 5

Elo, H. A. (1980) Comp. Biochem. Physiol. 67B, 221-224 Green, N. M. (1975)Adv. Protein Chem. 29, 85-133 Bayer, E. A. and Wilchek, M. (1978) Trends Biochem. Sci. 3, N257-N259 Bayer, E. A. and Wilchek, M. (1980) Methods Biovhem. Anal. 26, 1-45 Bayer, E. A., Wilchek, M. and Skutelsky, S. (1976) FEBS Lett. 68, 240-244 6 Papermaster, D. S., Schneider, B. G., Zorn, M. A. and Kraehenbuhl, J. P. (1978)J. CellBiol. 77, 196-210 7 Bayer, E. A., Skutelsky, E., Goldman, S. et al. (1983)J. Gen. Microbiol. 129, 1109-1119 8 Warnke, R. and Levy, R. (1980)J. Histochem. Cytovhem. 28, 771-776 9 Hsu, S.-M., Raine, L. and Fanger, H. (1981)J. Histochem. Cytochem. 29, 577-580 10 Hsu, S.-M. and Raine, L. (1981)J. Histochem. Cytochem. 29, 1349-1353 11 Berman, J. W. and Basch, R. S. (1980)J. ImmunoL Methods 36, 335-338 12 Berger, M., Gaither, T. A., Cole. R. M. el aL (1982) Mol. ImmunoL 19, 857-864 13 Bayer, E. A. Skutelsky, E. and Wilchek, M. (1979)Methods EnzymoL 62, 308-315 14 Berger, M., Brown, E., Cole, R. and Frank, M. M. (1982)Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 829 15 Becker, J. M. and Wilchek, M. (1972)Bioehem. Biophys. Acta 264, 165-170 16 Haimovich, J. and Sela, M. (1966)J. Immunol. 97, 338-343 17 Guesdon, J.-L., Ternynck, T. and Avrameas, S. (1979) J. Histochem. Cytochon. 27, 1131-1139

18 Guesdon, J.-L. and Avrameas, S. (1980) Ann. Immunol. (Part)) 131C, 389-396 19 Rappuoli, R., Leoncini, P., Tarti, P. and Neri, P. (1981)Anal Biochem. 118, 168-172 20 Wilcheck, M. (1980)J. Solid-Phase Biochem. 5, 193-195 21 Finn, F. M., Titus, G., Montibeller, J. A. and Hofman, K. (1980)J. BioL Chem. 255, 5742-5746 22 Nerurkar, L. S., Jacob, A. J., Madden, D. L. and Sever, J. L. (1983) , f Clin. Microbiol. 17, 149-154 23 Arnon, R. and Sela, M. (1982) ImmunoL Rev. 62, 5-27 24 Philpott, G. W., Kulezycki, A., Jr, Grass, E. H. and Parker, C. W. (1980)J. ImmunoL 125, 1201-1209 25 Urdal, D. L. and Hakomori, S. (1980)J. Biol. Chem. 255, 10509-10516 26 Bayer, E. A. and Wilchek, M. in Liposome Technology (Gregoriadis, G., ed.), CRC Press (in press) 27 Wood, G. S. and Warnke, R. (1981) J. Histochem. Cytochem 29, 1196-1204 28 Ling, N. R. and Kay, J. E. (1975) Lymphocyte Stimulation, 2nd Edn, Elsevier/North-Holland, Amsterdam 29 Wynne, D., Wilchek, M. and Novogrodsky, A. (1976) Biochem. Biophys. Res. Commun. 68, 730-739 30 Ravid, A., Novogrodsky, A. and Wilchek, M. (1978)Eur. J. Imrnunol. 8, 289-294 31 Berger, M. (1975) Biochemistry 14, 2338-2342 32 Wilchek, M., Spiegel, S. and Ravid, A. (1981) inMecham)ms of Lymphocyte Activation (Resch, K. and Kirchner, H., eds), pp. 19-30, Elsevier, New York 33 Spiegel, S. and Wilchek, M. (1981)J. Immunol. 127, 572-575 34 Heitzmann, H. and Richards, F. M. (1974)Proc. NatlAcad. Sci. USA 71, 3537-3541 35 Bayer, E. A. Skutelsky, E., Wynne, D. and Wilchek, M. (1976) J. Histovhem. Cytochem. 24, 933-939 36 Spiegel, S., Skutelsky, E., Bayer, E. A. and Wilchek, M. (1982)Biochim. Biophys. Acta 687, 27-34 37 Jawiewicz, M. L., Schoenberg, D. R. and Mueller, G. C. (1976) Exp. Cell Res. 100, 213-217 38 Costello, S. M. Felix, R. T. and Giese, R. W. (1979) Clin. Chem. 25, 1572-1580 39 Langer, P. R. Waldrop, A. A. and Ward, D. C. (1981) Proc. :VattAcad Sci. USA 78, 6633-6637 40 Korenman, S. G. and O'Matley, B. W. (1970) Methods EnzyrnoL 18A, 427-430 41 Friedman, A. and Cohen, I. R. Immunogenetics (in press) 42 Friedman, A., Zerubavel, R., Gitler, C. and Cohen, I. R. Immunogenetics (in press)

Antigenic variation in African trypanosomes: DNA rearrangements program immune evasion Marilyn Parsons, Richard G. Nelson and Nina Agabian Individual B cells express only one of the many variable-region genes of the VH gene repertoire. Likewise, individual African trypanosomes express only one surface-antigen gene of the large surface-antigen gene repertoire. In both kinds of cells, expression is controlled at the level of transcriptional activation and has been shown to involve rearrangement of genomic DNA. Here, Nina Agabian and her colleagues review recent studies on the molecular mechanisms controlling trypanosome suorace-antigen gene expression. The phenomenon of antigenic variation in African trypanosomiasis or sleeping sickness has fascinated parasitologists, immunologists and, more recently, molecular biologists since its description in 1909 by Paul Ehrtich 1. The waves of parasitemia characteristic of this chronic disease of man and his domestic animals were hypothesized to result from the successive proliferation of antigenically distinct trypanosome populations. As antibody to one antigen type neutralized those cells, the fraction of trypanosomes expressing a different antigen would survive and proliferate to elicit, in time, another antibody response. This hypothesis has been amply

Department of Biochemistry SJ-70, Washington, Seattle, WA 98195, USA.



confirmed in the subsequent decades of research. The molecules responsible for antigenic variation are a family of membrane glycoproteins ranging from 55 000 to 65 000 in molecular weight L At any one time, only one of these proteins is expressed and it covers the surface of the protozoan to the exclusion of all other antigens. This protein, termed the variant surface glycoprotein (VSG), is distinct and characteristic for each trypanosome variant antigen type (VAT); each V S G is encoded by a separate structural gene. V S G protein structure and synthesis have recently been reviewed s. In this review, we will discuss the recent work of several laboratories aimed at elucidating the molecular mechanisms which mandate the expression of a single gene from the vast VSG gene repertoire and which regulate the expression of these © 1984,ElsevierSciencePublisher~B.V.,Amsterdain 0167- 4919/841502.00