immunolog), today, December 1980

19Gillis, S., Scheid, M. and Watson, j. (198{))j . lmmunol. (in press) 20 Watson, J., Aarden, L.A., Shaw, .J. and Paetkau, V. (1979) J. lrrzmunol. 122, 1633 21 Smith, K.A., Gillis, S., Ruscetti, F.W., Baker, P.E. and McKenziel, D. (1979) Proc.N.Y. Acad. Sci. 332,423 22 Watson,J. and Mochizuki, D. (1980) Irnmunol. Rev. 51,355 23 Watson, J., Mochizuki, D. and Gillis, S. (1980) in ICN-UCLA Syrnp. Conlrol of Cell division and Dzfferentialim7 (D. Cunningham, E. Goldwasser and j. Watson, eds), Academic Press, New York 24 Smith, K.A., Gillis, S. and Baker, P.E. (1979) in 7he Molecular Basis of lrnmune (',ell Function (J.G. Kaplan, ed.), p. 223, Elsevier/North-Holland Biomedical Press, Amsterdam 25 Farrar, Jv]. and Fuller-Bonar,J. (1980) Fed. Proc.39, 3 26 Smith, K.A., Lachman, L.B., Oppenheim, J.J. and Favata, M.F. (1980)J. Exp. Med. 151, 1551 27 Larsson, E.L., Iscove, N.N. and Coutinho, A. (1980) Nature (London) 283, 664

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28 Gillis, S. and Mizel, S.B- (1980) J. lmmunol. (submitted for publication) 29 Baker, P.E., Gillis,S. and Smith, K.A. (1979)J. Exp. Med. 149, 273 30 yon Boehmer, H., Hengartner, H., Nabholz, M., Lernhardt, W., Schreier, M.H. and Haas, W. (1979) Eur. J. lmmunol. 9, 592 31 Rosenberg, S.A., Spiess, P,J. and Schwarz, S. (1978) J. [mmund. I21, 1946 32 Schreier, M.H. and Tees, R. (1980) Int. Archs. Allerg.y Appl. Immunol. 61,227 33 Gillis, S., Baker, P.E., Ruscetti, F.W. and Smith, K.A. (1978) .,7. Exp. Med. 148, 1093 34 Zarling,J.M. and Bach, F.H. (1979) Nature (London) 280, 685 35 Glasebrook, A.L. and Fitch, F.W. (1979) Nature (London) 278, 171 36 Bach, F.H., Inouye, H., tlank, J.A. and Alter, BJ. (1979) Nature (London) 281,307

Biochemistry of major human histocompatibility antigens Michael J. Owen and Michael J. Crumpton Imperial Cancer Research Fund Laboratories, P.O. Box No. 123, Lincoln's Inn Fields, London WC2A 3PX. The major histocompatibility region, designated H L A in man and H - 2 in the mouse, controls lhe expression of at least three groups of gene product - - serum complement components and two sets of highly polymorphic cell-surface anligens, the so called histocompatibility antigens. In this review Michael Owen and Michael Crumpton describe some recent insights into Ihe biochemical structure of H L A antzgens.

The primary function of the major histocompatibility region is immune regulation ~. The two groups of histocompatibility antigens are the classical major transplantation antigens (HLA A, B and C in man; H-2K, D and L in the mouse) and the I-regionassociated antigens (HLA-DRw in man; Ia in the mouse) (Fig. 1). The former are coded in h u m a n beings by genes located at the closely linked H L A A, B and C loci and are expressed on essentially all cell types. H u m a n la (HLA-DRw) antigens are most probably also coded by several loci coincident with or close to the HLA D(R) locus. In contrast to the major transplantation antigens, they have a much more restricted distribution, being differentiation antigens expressed primarily on B lymphocytes and monocytes. Biologically, major histocompatibility antigens function principally as regulators o f T-lymphocyte receptor and effector functions2. The major transplantation antigens participate in interactions between cytotoxic T lymphocytes and target ceils, whereas Ia antigens present foreign antigens to helper or suppressor T cells. Possession of particular antigens from both groups has been correlated with an individual's susceptibility to disease 3. Although this

review primarily describes results obtained with the h u m a n antigens, it is apparent, especially from studies in mice 4, that other vertebrate species possess structurally homologous gene products that mediate identical tunctions 5. The structure of the H L A A and B antigens is known in considerable detail (Fig. 2) - indeed, these antigens are among the most extensively characterized plasma-membrane glycoproteins. They comprise a polypeptide of molecular weight about 44,000, which is associated non-covalently with one of molecular weight 11,600, namely [32-microglobulin~'. The heavy chain represents the HLA A and B gene product, possesses a single asparagine-linked oligosaccharide unit, carries the allotypic determinants (i.e. those distinguishing members of the same species) and has been shown to be t r a n s m e m b r a n e in orientation, traversing the lipid bilayer once 7. About thirty amino acids at its carboxy-terminus form a hydrophilic domain, which is located at the cytoplasmic face of the cell-surface membrane; this domain is remarkable for its high serine content (8 out of 32 residues in HLA B7), at least one of which is phosphorylated s. [32-microglobulin is associated with the heavy chain on the cell surface and is not inserted in the hydrophobic part of © Elsevier/North-HolLand Biomedical Press 1980

118

immundogy today, December1980

H-2

I 0 . . . . . GLO . . . . .

I

K

1

I

C4(Ss,SIp,G)-

D,L

Q

TL-

ABJEC

centromore

1

0 . . . . . GLO . . . . . . . . . . . . . . . .

D(R) I

C4(Ch,R)C2,Bf HLA

B--C-

.A--]

Fig. 1. Schematic representation of the genetic maps of the mouse H-2 and human HLA regions.

In the mouse, K, D and L code for the major transplantation antigens; I represents the immune-response region which is divided into several sub-regions (A, B, J, E and C). In the human, B, C and A code for the major transplantation antigens and D(R) codes for the major antigenic determinants responsible for mixed lymphocyte responses. C2 and C4 refer to the C2 and C4 components of complement, Bfto factor B of the alternative complement pathway, Ch and R tothe Chido and Rogers blood groups, and GLO to gloxylase. the lipid bilayer of the cell-surface membrane 9. It is non-glycosylated, coded by a gene on a chromosome (No. 15) different from that which carries the H L A region and is non-polymorphic in man. Of special interest is the striking homology between the aminoacid sequence of 132-microglobulin and the C3H (third constant region of the heavy chain) domain of immunoglobulin G. H L A C antigens are present at about 10°7o of the amount of the H L A A and H L A B antigens but although they have a grossly similar structure they have not been extensively characterized. In contrast to the H L A A, B and C antigens, H L A D R w antigens (Fig. 3) comprise two polypeptide chains of molecular weights about 34,000 and 28,000 [designated u(p34) and 13(p28) respectively] 6,~. Both chains are glycosylated and are tightly associated, albeit non-covalently. Both polypeptides span the plasma membrane 7. The a chain is phosphorylated a t its carboxy-terminal region, although the level of phosphorylation appears to be considerably less than for the H L A A and B antigen heavy chain. A complete understanding of the biological functions of the H L A A, B and D R w antigens relies upon an intimate knowledge of their molecular structures including their three-dimensional conformations. Recent work has revealed some probable structural domains as well as regions that may be involved in cellular recognition. The remainder of this review is concerned with these and other biochemical aspects that are pertinent to structure-function relationships. HLA A and B antigens

Structural domains, evolutionary considerations and functional determinants. The complete amino-acid sequence of the papaincleaved, extracellular portion of the H L A B7 antigen has been determined (Fig. 4) 1°. Most interestingly, this sequence shows significant homology with that of

immunoglobulin. Thus, the papain-cleaved fragment contains two disulphide-bonded loops (Fig. 2). The first of these is similar in size (63 amino-acid residues), but shows no detectable sequence homology with the disulphide loop of an immunoglobulin variable/ constant region domain. The sequence of the second disulphide loop (86 residues) is, however, judged statistically to resemble that of an immunoglobulin constant region, suggesting that this portion of the HLA-B7 polypeptide chain is potentially able to fold into an immunoglobulin domain-like structurC 1. This result, when taken together with the close structural similarity between ]32-microglobulin and immunoglobulin, argues strongly that the major transplantation antigens and immunoglobutin have a common evolutionary origin, possibly in the role of primordial recognition molecules 12. Of special interest in this respect is the observation that the Thy-I antigen (a rodent cell-surface differentiation glycoprotein of molecular weight about 25,000) also shows significant homology to immunoglobulin with respect to aminoacid sequence, arrangement of intrachain disulphide bridges and [3-pleated sheet conformation I~. Conceivably, this surface-membrane glycoprotein also functions in celt-cell recognition and interaction. Comparison of the H L A B7 sequence with partial sequence data for H L A A and H-2K molecules 4,~° has revealed two clusters of variable amino-acid residues, nos 65-80 and 105-144. One or both of these regions is (are) most probably responsible for the antigens' extensive serological polymorphism (i.e. they include the alloantigenic determinants). They are likely to be functionally important as well, although the way in which they mediate function has yet to be designated. Direct evidence in support of these proposals comes from the study of H-2" and H-2 d mice with mutations in their H-2K a n d / o r H-2D gene products ~4. In several mutants displaying altered immunological reactivities, including rejection of skin grafts, amino-

immunology loday, December 1.980

119

acid substitutions were located in the above regions of increased variability, suggesting that these regions are indeed involved in m e d i a t i n g T - l y m p h o c y t e dependent reactions. A picture has thus emerged from the comparative sequence studies of the extracellular portion of H L A A and B antigens (similarly, H-2K and D antigens) of four independent structural domains (Fig. 2). 132microglobulin forms one domain and the other three are located on the papain-cleaved fragment of the heavy chain. One of these domains (%) is highly conserved amongst H L A A and B antigens and is structurally homologous to both the 132-microglobulin and immunoglobulin constant-region domains. The clusters of sequence variation (Fig. 4) are located on the a~ and a 2 domains, but it is conceivable that the folding of the polypeptide chain brings these clusters together to form a single alloantigenic site. Although the functional relevance of the individual domains is unknown, each domain may regulate a different aspect of the antigens' function. ALLOANTIGENIC

Bioaynthesis Studies of H L A A and H L A B antigen biosynthesis have indicated that it proceeds via a sequence of steps similar to those observed for various secreted and virus-membrane glycoproteins 1~. One aspect of their biosynthesis, namely the regulation of antigen expression at the cell surface is, however, potentially of considerable functional importance. Cells from the Burkitt lymphoma celMine Daudi do not express H L A A or B antigens at their surface and do not synthesize detectable amounts of 132-microglobutin. The H L A A and B gene products are, however, present intracellularly, but are transported slowly, if at all, from their site of synthesis 8,~5. One explanation for the absence of surface expression of the antigens is that association of 132-microglobulin with the H L A A and B gene products is an essential prerequisite for intracellular processing. In other words, 132-microglobulin regulates the surface expression of the antigens: other explanations are, however, possible (see below). This way of regulating the cell-surface structure (i.e. cell function) may not be unique to H L A A and B antigens. For instance, it appears possible that the surface expression of membrane-associated immunoglobulin by pre-B-lymphocytes is controlled in a similar way by free immunoglobulin L chain.

Interaction with virus proteins

m

A

)LASM

Fig. 2. Schematic representation of an HLA A or B m o l e c u l e in the plasma m e m b r a n e . The molecule is viewed as comprising four domains at the external surface of the lipid bilayer. 132-microglobulin comprises one domain; the ¢*~,% and % domains are formed by the heavy chain. A hydrophobic stretch of the heavy chain traverses the lipid bilayer of the plasma membrane. The C-terminal, hydrophilic peptide is exposed on the cytoplasmic face of the membrane. Digestion of the plasma membrane or detergent-solubilized HLA A and B antigens with papain produces a water-soluble fragment of apparent molecular weight 34,000 which retains alloantigenicity. Reproduced from Strominger el at., in: Currenl Topics in Devet@mentat Biology, Devet@menlat Imm~mology, Vol. 14, Academic Press, New York (in press).

The killing of virus-infected cells by cytotoxic T cells depends on a self-recognition mechanism involving, in mice, H-2K a n d / o r D (i.e. in man H L A A and/or B) determinants 2. A possible mechanism for such restricted killing is the recognition by the killer cell of viral antigen in association with the major transplantation antigen (the 'alteJ-ed-self' model). Little persuasive evidence in support of this hypothesis has emerged, although an association of rat major transplantation antigens with an adenovirus protein has been reported 16. One problem of probing the mechanism may be the weak affinity of killer-target cell interaction Iv, in which case the use of purified viral and major transplantation antigens incorporated into liposomes (artifical lipid bilayers) may help. H L A A and B antigens, when inserted into liposomes, interact specifically with Semliki Forest virus (SFV) spike glycoproteins ~. It was suggested that the transplantation antigens act as virus receptors, since antigencontaining liposomes competed for binding of virus to cells; also Daudi cells, which do not express H L A A and B antigens, were resistant to virus infection. The fact that various DNA and RNA viruses, including SFV, replicate in cells apparently lacking maior transplantation antigens suggests, however, that such antigens are unlikely to be the universal receptor for viral penetration and infection ~9. The explanation for the resistance of Daudi to SFV infection is, therefore, unclear. It is, however, possible that an as-yet, unknown lesion is responsible both for the lakk of sur-

120 face expression of the H L A A and B antigens and for the resistance to virus infection. An intriguing possibility is a defect in intracellular processing, responsible both for transport of H L A A and B antigens to the cell surface and of infective virus from the surface to the cell interior. HLA DRw antigens

Mouse Ia antigens are divided into two groups, depending on whether they are coded by the I-A or IE / C subregions of the I region (Fig. 1) l. H L A D R w antigens are the h u m a n counterparts of mouse Ia antigens and are coded by the H L A D ( R ) locus or an adjacent locus 4,6,s. Although the currently designated h u m a n antigens are homologous with the mouse IE / C subregion Ia antigens, the counterparts of the I-A subregion antigens most probably also exist. Ia antigens are the p r i m a r y stimulators of mixed lymphocyte responses. More importantly, in association with foreign antigen either on the cell surface or as 'soluble factors', they mediate interaction with Thelper lymphocytes 2. Structure and polymorphism The structure of H L A D R w antigens is represented schematically in Fig. 3. Their detailed structural characterization has lagged considerably behind that of the H L A A and B antigens, primarily because of difficulties in identifying and separating the different subregion products and in isolating the separate c~(p34) and [3(p28) polypeptides. In mice, amino-acid-sequence studies have been restricted to the N-termini but when considered in conjunction with peptide 'fingerprints' they have led to several concepts 2°. (Studies of the H L A D R w antigens have yielded similar results but the data are less extensive.) Firstly, the a and [3 chains are structurally distinct. Secondly, the a chains of I-A subregion antigens differ from those of the I - E / C subregion and similarly for the [3 chains. Thirdly, the 13 chains of different haplotypes show considerable structural variation, indicating that these chains most probably mediate the polymorphism and are coded by an I-region gene. Some structural variation has, however, been reported for the a chains, also suggesting that their expression is similarly controlled by the I-region. Fourthly, independent evidence that a and 13 chains are encoded by the I region has come from studies of mice with intra-I-region recombinations. These studies further suggest that the a chains m a p to the respective 1-A or I - E / C subregions (as expected) but that the [3 chains of both the I-A and I - E / C sub region antigens map to a site in the I-A locus 21,22. Although the simplest interpretation of the above results is that the structural genes encoding the a and [3 chains are both included in the major histocomparability region, it is not possible from classical genetics to distinguish between structural and c o n -

immunologytodoy, December197,0

trolling genes. In this respect it may be significant that the chains of other oligomeric proteins comprising non-identifiable polypeptides are encoded by genes on different chromosomes. An important question is whether polymorphism is mediated by the carbohydrate or polypeptide moieties. Arguments in support of the view that Ia alloantigenicity resides in the polypeptide include heat and protease sensitivity and the observation that alloantigenicity survives depletion of carbohydrate by glycosidases or tunicamycin. It has, however, been claimed that mouse serum contains glycolipid(s) w h i c h i n h i b i t ( s ) Ia a l l o a n t i s e r a a n d w h o s e a n t i g e n i c i t y is d e s t r o y e d by n e u r a m i n i d a s e or periodate but not by pronase >. The suggestion that Ia alloantigenic determinants reside in the carbohydrate is highly contentious and the above results should be interpreted with caution until they have been confirmed in other laboratories. If true, however, they imply that some genes in the I region encode, or control the expression of, glycosyl transferases. An invariant ttLA DRw-associated polypeptide In addition to the a and [3chains detected by sodium

o(

NH.

,B

NH2,

C ....

Structurally variable; atlotypic

site?

papain

plasma membrane

~COOH P04)

cytoplasm

Fig. 3. Schematic representation of an HLA DRw molecule in the plasma membrane.

The molecule is viewed as comprising two chains tightly associated non-covalently, the c~chain carries two asparagine-linked oligo-saccharide units, spans the lipid bilayer and is phosphorylated at the carboxy-terminus; the [3chain carries on asparagine-linked oligo-saccharide unit and also spans the bilayer. The main structural polymorphism and, therefore, probably the alloantigenic determinant resides in the [3chain.

zmmunology led©,, December 1980

121

lO 20 30 40 50 60 70 80 90 HLA-B7 NH2-GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTORESLRNLRGYYNQSEA HLA-A2

NH2---F

H2-Kb

NH2--P--L--V=A

A, L~YME

Z-M--E

KV--H-H-~-VD-GT

EN--y--R=M

E-E--KA=GN E-SF-VD~T= L

KS

HLA-B7

lO0 llO 120 130 140 150 160 170 ~30 GSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYI ALNEDLRSWTAADTAAQI TQRK~IEAAREAEQRRAYLEGECVEWLRRYLENGKDKLE

HLA-A2

- - R - -

H-2Kb

--I-

HLA-B7

190 200 210 220 230 240 250 260 270 RADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQROGEDQTQDTELVETRPAGDRTFEKWAAVVVPSGEEQRYTCHVQHEGLPKPLT

--~2~-W-F - - Y H

K

H-2Kb

M

KH

V

M-- L - - ~ - - Q - G - -

M

T-RL

n_ ~S

T

- - c -t 7 -

-K

¥-

Fig. 4. Comparison of the complete primary sequence of HLA B7 antigen (papain-cleaved fragment) with HLA A2 and H-2K b antigens. Amino acid residues in HLA A2 and H-2Kb which are homologouswith HLA B7 are indicated by the solid line. Amino-acidresidues are indicated by the single letter code: A, ala; B, asn/asp; C, cysteine;D, asp; E, glu; F, phe; G, gly; H, his; I, ile; K, lys; L, leu; M, met; N, asn; P, pro; Q, gin; R, arg; S, ser; T, thr; V, val; W, trp; Y, tyr; Z, gln/glu. Reproduced from Ref. 10.

dodecylsulphate-polyacrylamide gel analyses of immunoprecipitates from [~5S] methionine-labelled cells, two-dimensional gel analyses have revealed a third polypeptide of molecular weight about 31,000 (p31)24.2~. This chain was first detected as an invariant basic spot in all mouse Ia immunoprecipitates of the IA and I - E / C subregions. More recently it has been observed also with h u m a n HLA DRw antigens. Peptide maps and two-dimensional gel electrophoresis indicate that the basic spot is distinct from the c~and [3 chains, but have provided no clue as to the relationship of this invariant polypeptide to the major histocompatibility region. Subcellular fractionation studies of biosynthetically labelled h u m a n B lymphoblastoid cells demonstrate that the 31,000 molecular weight component is restricted to intracellular membranes and that the plasma-membrane-located HLA DRw antigen comprises a and [3 chains only. These results do not, however, rule out the possibility that a processed, more mature form of the basic spot of 34,000 molecular weight is associated with the HLA DRw antigens at the cell surface. The function of the invariant polypeptide can at present only be guessed at. In man it might be related to an HLA D(R) subregion product. One intriguing possibility is that it controls the expression of the a and [3chain complex at the cell surface, perhaps by interacting with cytoskeletal components or an intracellular transport pathway. Conclusions Considerable progress has been made in the detailed structural characterization of H L A A and B antigens and there is every likelihood that their threedimensional structure will be described in the near

future. The biochemistry of H L A - D R w antigens has lagged behind, partly because the H L A - D ( R ) region probably has several subregions. In this respect, a more detailed genetic analysis of the HLA D(R) region would undoubtedly promote biochemical progress. Two major gaps in our knowledge of H L A A, B, and C, and HLA DRw antigens are the precise relationships between their structures and functions, and the organization of their genes in the major histocompatibility region. Gene organization is currently being explored with recombinant DNA techniques. These studies should provide unambiguous answers to many current speculations: e.g. are there 'variable' and 'constant' gene segments as in immunoglobulin genes? Is the information for all allotypes included in the genome or are the major histocompatibility region genes truly altelic? The availability of cells expressing cloned genes and of large amounts of the purified antigens will prove invaluable in studies of structure-function relationships. However, progress in this field will depend to a large extent on the availability of cloned functional T-lymphocyte cell lines (i.e. antigen-specific killer and helper cells). References 1 Barnstahle, C. J., Jones, E. A. and Bodmer, W. F. (1979) Int. Re~,. Biochem. 22, 151-225. 2 Zinkernagel, R. M. and Doherty, P. C. (1979) Ad~,. lmmunol. 27, 51-177 3 Dausset, J. and Svejgaard, A. (eds) (]977) HLA and Disea.~e Munk,s~aard, Copenhagen

4 Nathenson, S. G., Ewenstein, B. M., Uehara, H., Martinko,J. M., Coligan,J. E. and Kindt, T. J. (1980) in Curren! Tren& in Histocompalibili(;' (Ferrone, S. and Reisfeld, R., eds), Plenum Press, New York

immunology today, December7980

122 5 G6tze, D. (ed.) (1977) The Mqlor HistocompatibilitySystem in Man and Ammals, Springer-Verlag, Berlin 6 Barnstable, C. J., Jones, E. A. and Crumpton, M.J. (1978) Br. Med. Bull. 34, 241-246 7 Walsh, F. S. and Crumpton, M.J. (1977) Nature (London) 269, 307-311 8 Strominger, J. L. el al. in The Role of Major Histocompatibility Compex in Immunobiology (Benacerraf, B. and Doff, M. E. eds), Garland Press Publishing Inc., New York (in press) 90wen, M. J., Knott, J. C. A. and Crumpton, M. J. (1980) Biochemistry 19, 3092-3099 10 Orr, H. T., Lopez de Oastro, J. A., Parhana, P., Ploegh, H. L. and Strominger, J. L. (1979) Proc. Natl. Acad. Sei. U.S.A. 76, 4395-4399 11 Strominger, J. L., Orr, H. T , Parham, P., Ploegh, H. L., Mann, D. L., Bilofsky, H., Saroff, H. A., Wu, T. T. and Kabat, E. A. (1980) Scand. J. hnmunol. 11,573-592 12 Bodmer, W. F. (1972) Nalure (London) 237, 139-145 and 183 13 Campbell, D. G., Williams, A. F., Bayley, P. M. and Reid, K. B. M. (1979) Nature (London) 282, 341-342 14 Nairn, R., Yamaga, K. and Nathenson, S. G. Annu. Rev. Genet. Vol. 14 (in press) 15 Owen, M. J., Kissonerghis, A.-M. and Lodish, H. F. J. Biol. Chem. (in press.)

16 Kvist, S., Ostberg, L., Persson, H., Philipson, L. and Peterson, P. A. (1978) Proc.Natl. Acad. Sci. U.S.A. 75, 5674-5678 17 Honeycutt, P.J. and Gooding, L. R. (1980) Eur. J. Immunol. i O, 363-370 18 Helenius, A., Morein, B., Fries, E., Simons, K., Robinson, P., Schirrmacher, V., Terhorst, C. and Strominger, J. L. (1978) Proc.Natl. Acad. Sci. U.S.A. 75, 3846-3850 19 Oldstone, M. B. A., Tishon, A., Dutko, F. J., Kennedy, S. I. T., Holland, J. J. and Lampert, P. W. (1980) J. Virol. 34, 256-265 20 Uhr, J. W., Capra, J. D., Vitetta, E. S. and Cook, R. G. (1979) Science206, 292-297 21 Jones, P. P., Murphy, D. B. and Mcl)evitt, H. O. (1978) J. Exp. Med. 148, 925-939 22 Cook, R. G., Vitetta, E. S., Uhr, J. W. and Capra, J. 1). (1979) J. Exp. Med. 149, 981-986 23 Parish, C. R., Chilcott, A. B. and McKenzie, I. F. C. (1976) Immunogenetics3, 113-128 24 Jones, P. P., Murphy, D. B., Hewgill, D. and McDevitt, H. O. (1979) Mol. Immunol. I6, 51-60 25 Charron, D. J. and McDevitt, H. O. (1979) Proe.Natl. Acad. Sei. U.S.A. 76, 6567-6571

(te(hniques New methods of analysing for antigens and glycoproteins in complex mixtures Brian H. Anderton

and

Department of Immunology, St. George's Hospital Medical School, London, SW17 ORE

Robin C. Thorpe National Institute tbr Biological Standards and Control, London NW3 6RB

The analysis of complex mixtures of proteirzs and particularly those which make up macromolecular assemblies including membranes, received a ma.]or Jillip in the late 1900s with the introduction of sodium dodecyl sulphate polyaerylamide gel electrophoresis ( SDS-PAGE). The resolving power of electrophoresis in polyacryIamide gels is considerably superior to that achieved with any other support medium including agar, the immunochemist's standby. The essential physical property which makes polyacrylamide the medium of choice is the limiting pore size of the gel: its sieving effect on the movement of macromolecules and the reduction of diffusion together result in increased sharpness of bands, On the other hand, in all the standard immunochemical techniques such as immunodiffusion, immunoelectrophoresis, and rockets, free diffusion of prolein molecules in the support medium is essential and agar or agarose satisfy this requirement. There has therefore been a search .for ways in which polyaerylamide gel electrophoresis, particularly SDS-gel electrophoresis of aggregates of proteins (as in membranes, viruses, the cellular cyloskeleton), can be adapted to immunochemical analysis of their constituent antigens. Here Brian Anderton and Robin Thorpe discuss several of the methods now in use. T h e s e m e t h o d s fall into two categories: those which rely u p o n c o m b i n i n g separation of antigens by S D S P A G E with electrophoresis or diffusion of antigen from the p o l y a c r y l a m i d e gel into a n t i b o d y - c o n t a i n i n g

agarose; and those in which a n t i b o d y is applied directly to the surface of the gel or to a p a p e r transfer of the gel (these are p r i m a r y b i n d i n g assays). It is the latter technology which is now proving to be so useful