Isolation and partial characterisation of a 26 kilodalton antigen from Plasmodium falciparum recognised by an inhibitory monoclonal antibody

Molecular and Biochemical Parasitology, 29 (1988) 125-132 Elsevier

125

MBP 00972

Isolation and partial characterisation of a 26 kilodalton antigen from Plasmodium falciparum recognised by an inhibitory m o n o c l o n a l antibody Ranjan Ramasamy 1, Richard J. Simpson 2, Annette Dexter 1, Marissa Keeghan 1, Carol Reed 1, Gillian Bushell 1., Leanne T. Ingram 1, Thrift Henderson 1, Margaret B. Moloney 3, Robert L. Moritz 2, Michael R. Rubira a and Chev Kidson 1 1Queensland Institute of Medical Research, Brisbane, Queensland, Australia, 2Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, and 3Cornnrnonwealth Serum Laboratories, Parkville, Victoria, Australia (Received 23 November 1987; accepted 27 January 1988)

A 26 kDa protein, present in trophozoites and schizonts of Plasmodium falciparurn, has been identified as the target of a monoclonal antibody that weakly inhibits parasite growth in vitro. The antigen has been purified to homogeneity by immuno-affinity chromatography and electrophoresis. The sequence of 19 amino acids at the N-terminus of the protein has been determined. Key words: Amino acid sequence; Antigen isolation; High performance liquid chromatography; Malaria; Monoclonal antibody; Plasrnodium falciparum

Introduction

There is considerable interest at present in producing a molecular vaccine against falciparum malaria based on the asexual blood stages of Plasmodium falciparum. Critical to this process is the identification of parasite molecules that are capable of evoking a protective immune response in man. Monoclonal antibodies (Mabs) and monospecific polyclonal antibodies have proved to be useful for this purpose [1]. The target antigens of several Mabs that inhibit the growth of the asexual blood stages in vitro have been characterised. These include merozoite surface [2,3] and rhoptry [4] antigens. It is likely that merozoite *Present address: School of Science, Griffith University, Nathan 4111, Australia Correspondence address: R. Ramasamy, Queensland Institute of Medical Research, Bramston Terrace, Brisbane, Queensland 4006, Australia. Abbreviations: Mab, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RIPA, radio-immune-precipitation buffer; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid.

surface molecules are protective because of their accessibility to antibodies and immune effector cells of the host. This is supported by the ability of purified or recombinant merozoite surface proteins to confer protection to immunised monkeys [5,6]. The contents of the rhoptries are extruded from the merozoites during the invasion of red cells and may play a role in the invasion process [7]. This can explain the inhibition of parasite development observed with antibodies to the rhoptry antigens of P. falciparum in vitro [4]. We report here the identification, from the asexual blood stage of P. falciparum, of a 26 kDa antigen that is the target of a Mab that weakly inhibits parasite growth in vitro. The Mab has been used to isolate the antigen by immuno-affinity chromatography and gel electrophoresis for partial N-terminal amino acid sequence determination. Materials and Methods

Parasite material. The FCQ-27 isolate, clone D10 of P. falciparum, originating in Papua New Guinea [8], was cultured in A ÷ erythrocytes (for

0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

126 small scale cultures) or O + erythrocytes (for bulk cultures at the Commonwealth Serum Laboratories) according to the method of Trager and Jensen [9]. Parasite growth was synchronised by sorbitol treatment [10] for isolating preparations of the mid-ring, trophozoite and schizont stages. Late stage parasites were isolated by gelatine sedimentation [11] at the trophozoite stage and then labelled with [35S]methionine, [63H]glucosamine or [3H]myristic acid as reported elsewhere [12-14]. Immunofluorescence on late stage FCQ-27 parasites was performed essentially as previously described [13]. Briefly, thin smears of late stage parasites on slides, fixed in a 9:1 mixture of acetone and methanol for 30 rain at -20°C, were reacted with undiluted Mab 1Cll53 (hybridoma culture supernatants) for 16 h at 4°C in a humid chamber. The slides were then washed in 0.01 M phosphate-buffered saline, pH 7.2 (PBS) and reacted with a 1:20 dilution of fluorescein conjugated affinity purified rabbit antibodies to mouse immunoglobulins (Dako, Denmark) for 3 h at 37°C. Finally, the slides were washed and viewed with incident ultra-violet illumination in an Olympus microscope. Photographs were taken on Ilford XP1 film with a 2 min exposure. The parasites were then stained with Giemsa and cells corresponding to those photographed for immunofluorescence were located with the help of markings on the slide and rephotographed. Immunofluorescence tests on other parasite isolates to determine the strain variation of the epitope were carried out in a similar manner.

In vitro growth inhibition assay. This was performed essentially as described previously [4]. The IgG1 mouse Mab 1Cll-53 was produced by standard procedures [4]. The IgG fractions of the rabbit polyclonal antiserum (described below), absorbed 3× with human A + erythrocytes, and the ascites fluid from mice carrying the hybridoma were isolated by binding to protein A-Sepharose (Pharmacia, Piscataway, N J) and eluting with 0.1 M glycine-HC1 buffer pH 2.6. The eluted antibodies were neutralised, dialysed exhaustively against RPMI 1640 medium and then sterilised by passing through a 0.22 Ixm surfactant-free filter. The antibodies were then added at differ-

ent concentrations to replicate cultures of FCQ27 in 96-well tissue culture plates. Control cultures contained no added antibody. The wells thus finally contained malaria culture medium with a 5% haematocrit and approximately 1% late trophozoite parasitaemia, and varying concentrations of Mab or polyclonal antibodies. The cultures were incubated until after reinvasion had occurred and then 3 ~xCi of [3H]hypoxanthine (Amersham, U.K.) was added in 100 t~1 of fresh RPMI 1640 medium per well. The cultures were then maintained until the parasites matured to schizonts. The cells were harvested onto Whatman glass fibre discs and washed successively with ice-cold 10% trichloroacetic acid (TCA), 5% TCA and 95% ethanol. The discs were then dried, immersed in toluene scintillant and radioactivity determined in a Packard scintillation counter.

Immune-precipitation and Western blotting procedures. These were performed as described elsewhere in detail [13,15]. Briefly, for immune-precipitation, 50 I~l of radioactively labelled packed parasitised erythrocytes at more than 50% parasitaemia were lysed in 1 ml of low salt radio-immune-precipitation buffer (RIPA: 10 mM TrisHC1 pH 7.4 containing 1% deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulphate (SDS), 20 mM E D T A and 150 mM NaC1) and insoluble material removed by centrifugation at 100 000 × g for 20 min. Immuno-absorbent beads were prepared by incubating 50 I~1 of protein ASepharose (Pharmacia, Piscataway, N J) with 100 ~xl of 1Cll-53 ascites for 2 h with mixing at 4°C. Protein A-Sepharose incubated with unrelated Mabs were used as controls. 0.5-1 ml of radiolabelled parasite lysate was incubated with the immuno-absorbent beads for 2 h at 4°C to bind specific antigen. The beads were washed twice in low salt RIPA buffer, once in high salt RIPA (RIPA containing 1.5 M NaC1) and finally in distilled water. The bound antigen was extracted with Laemmli sample buffer [16] and analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) [16]. For Western blotting, 5 p~l of parasitised erythrocytes containing midrings, trophozoites or schizonts at 8% parasitaemia, dissolved in 100 I~1 of 2 × concentrated Laemmli sample buffer were subject to SDS-PAGE and

127 transferred to a nitrocellulose membrane. Unparasitised erythrocytes were used as a control. After blocking unreacted sites on the membrane with 5% non-fat milk powder in PBS for 16 h at 4°C, the membrane was incubated with a 1:100 dilution of the rabbit polyclonal antiserum (described below) for 3 h at 22°C. The membrane was washed and incubated with 125I-protein A (specific activity >1.1 GBq mg -1, Radiochemical Centre, Amersham, U.K.) at 10 kBq m1-1 for 1 h at 22°C. 5% non-fat milk powder was used as a diluent for reagents throughout the Western blotting procedure. The washed and dried blot was autoradiographed with Kodak X A R film using Dupont Lightning plus intensifying screens.

Affinity purification of antigen and the production of polyclonal antiserum. An affinity column was prepared by incubating 5 ml of protein A-Sepharose beads with 10 ml of 1Cll-53 ascites fluid. The antibody and Protein A were then covalently cross-linked with dimethyl pimelimidate [17]. 5 ml of parasites, >70% parasitaemia and composed of trophozoites and schizonts, were dissolved in 100 ml of low salt RIPA and passed through the column at 0.5 ml min -1. The beads were washed in low salt RIPA and the bound antigen eluted with 50 mM diethylamine, pH 11.5 containing 0.05% sodium deoxycholate and immediately neutralised with 1 M sodium dihydrogen phosphate. The eluted antigen was further separated by SDSPAGE and the 26 kDa band corresponding to the antigen cut out from the Coomassie stained gel. The protein was electro-eluted from the gel using an ISCO electro-elution apparatus (ISCO, Lincoln, NE). A rabbit was injected intra-muscularly with 5-10 p,g of purified protein in complete Freund's adjuvant and boosted after 4 weeks with an equivalent amount of protein in incomplete Freund's adjuvant. A third injection in PBS was given intra-muscularly after a further 4 week interval and the animal bled for serum 10 days later.

Recovery of antigen for sequencing. The 26 kDa antigen, affinity purified as described earlier, was separated by SDS-PAGE on 3 mm thick gels using highly purified reagents as described [18]. The Coomassie-stained protein was electroeluted from the gel. The pure antigen was recovered from the

SDS-PAGE electroeluate and freed of SDS and gel-related contaminants using inverse-gradient high-performance liquid chromatography (HPLC) as described [19]. Briefly, the electroeluate (200 p~l) was diluted to 1.5 ml with n-propanol in a sample loading syringe, thoroughly mixed and applied to an ODS-Hypersil column (5 Ixm particle size, 12 nm pore size, dimethyloctadecylsilica support, 100 x 2.1 mm internal diameter column, Hewlett Packard Co., Waldbronn, F.R.G.) which had been previously equilibrated with 90% n-propanol/10% water (Solvent A). Under these conditions the antigen was retained by the support while SDS and gel-related contaminants were washed through the column. The antigen was recovered by subsequently developing the column with a steep linear gradient (0-100% solvent B in 0.1 min). Solvent B was 50% n-propanol/50% water containing 0.4% (v/v) trifluoroacetic acid (Pierce Chemical Co., Rockford, IL). The flow rate used was 40 txl min -1 and the column temperature was 25°C. Chromatography was performed on a Perkin Elmer model LC4 liquid chromatograph equipped with a variable wavelength spectrophotometer (model LC95) and a fluorimeter (model LS4). Protein was detected by absorbance at 278 nm and by endogenous fluorescence using excitation and emission wavelengths of 280 nm and 260 nm, respectively.

Amino acid sequence analysis. Automated Edman degradation of the antigen was performed using an Applied Biosystems Model 470A gasphase sequenator as described [20]. Polybrene was used as the carrier [21].

Silver staining. The homogeneity and quantity of purified 26 kDa antigen was determined by silver staining with bovine serum albumin, ovalbumin and ot-chymotrypsinogen as standards. 1/100th of the isolated protein, together with known amounts of standard proteins, were separated by SDSPAGE in a minigel apparatus (Biorad, Richmond, CA) and the gel was silver stained as described by Heukeshoven and Dernick [22]. Results

Inhibition of in vitro growth of parasites. Visual examination of Giemsa stained parasite cultures

128 TABLE I In vitro growth inhibition by Mab 1Cll-53 Incubation

cpmxl0 -3 [3H]hypoxanthine incorporated _+ standard deviation

% inhibition

Expt. 1 Control (no antibody) Mab 1Cll-53 (25 Ixg ml 1) Mab 1Cll-53 (250 Ixg m1-1) Rabbit IgG antibodies (650 Ixg m1-1)

198_+2 168_+40 149_+12 197_+1

15 (ns) 25 (P<0.05) 0.5 (ns)

Expt. 2 Control (no antibody) Mab 1Cll-53 (750 Ixg ml

888_+23 557_+7

37 (P<0.001)

1)

The results are expressed as mean radioactivity incorporated in three (Expt. 1) or four (Expt. 2) replicate cultures _+ standard deviation of the mean. Probability was determined by Student's t-test allowing for unequal variances where appropriate.

on day 2 after addition of Mab 1Cll-53 showed fewer mature parasites compared with controls without antibody. Mab 1Cll-53 consistently inhibited parasite growth as measured by [3H]hypoxanthine incorporation. 250 Ixg m1-1 and 750 ixg ml 1 of Mab produced 25% and 37% inhibition respectively in different experiments (Table I). However, no inhibition was observed with up to 650 ixg ml-1 of the IgG fraction of the rabbit antiserum to the 26 kDa protein (Table I). A number of other anti-parasite Mabs did not inhibit in the assay and served as additional controls (data not shown).

Local&ation of the antigen in the parasite. Mab 1Cll-53 reacted with fixed trophozoite and schizont stage parasites giving a diffuse fluorescence pattern (Fig. 1). No fluorescence was seen in ringstages. The fluorescence seen in schizonts was clearly different from the grape pattern seen with monoclonal antibodies against the merozoite surface [23] or the paired, punctate fluorescence seen with antibodies against rhoptry antigens [4]. A comparison of the fluorescence and Giemsa staining of individual schizonts showed that the antigen was largely confined to the parasite inside the red cell (Fig. la and b). Within the resolution of light microscopy it was not possible to determine whether the antigen was in the parasite cytoplasm, parasitophorous vacuole or both. Occasionally spots located away from the main body of fluorescence were observed, suggesting that the antigen may also be found in vesicular structures

in the red cell cytoplasm (arrowed in Fig. lf). Antigen was often seen outside ruptured schizonts and appeared to be associated with merozoites (Fig. lc, fluorescence; Fig. ld, Giemsa stained parasites). After absorption with human A + red cells, to remove anti-red cell antibodies, the rabbit antiserum to the 26 kDa antigen gave the same fluorescence pattern, although with a greater intensity than Mab 1Cll-53 (Fig. le). In addition to FCQ-27, Mab 1Cll-53 reacted with all of the following laboratory maintained isolates that were tested by immunofluorescence: K1 (Thailand), FCR-3 (The Gambia), NF7 (Ghana) and V1 (Vietnam). When Mab 1Cll-53 was used for immuno-electron microscopy no reaction with parasite structures was seen under conditions where rhoptry and merozoite surface antigens were identified with other monoclonal antibodies (unpublished data).

Immune-precipitation and Western blotting of the antigen. Mab 1Cll-53 immune-precipitated a prominent 26 kDa [35S]methionine labelled protein, from radiolabelled parasite lysates (Fig. 2). None of the other Mabs used as controls precipitated this protein. At least one major band of this size is labelled with [35S]methionine in whole extracts of the FCQ-27 isolate [24]. The Mab 1Cll53 did not immune-precipitate a radioactively labelled antigen from [3H]glucosamine or [3H]myristic acid labelled parasite lysates under conditions where glycosylated and myristilated parasite proteins [25] are immune-precipitated

129

reaction was seen with pre-immunisation serum from the same rabbit. When equivalent numbers of mid-ring, trophozoite and schizont stage parasites in Western blots were probed with the rabbit antiserum, strong reactions were seen with the trophozoite and schizont preparations (Fig. 3). N-terminal amino acid sequence determination. !~iii?~ ¸

~

Silver staining of an aliquot of the 26 kDa antigen isolated by affinity chromatography and SDSPAGE separation showed that about 1 nmol (26 ~g) of the antigen was obtained, free of contamination with other proteins, from 5 ml of packed parasitised erythrocytes with >70% parasitaemia. A single peak of protein was seen when the antigen was further purified by HPLC to separate it from SDS, Coomassie blue and other gel-related contaminants that interfere with sequencing

4m

Fig. 1. Immunofluorescence staining of late stage parasites. (a) Fluorescence showing of Mab 1C11-53 with a schizont that was subsequently stained with Giemsa (b). (c) Fluorescence reaction with Mab 1Cll-53 in a mature parasite and in the region of merozoites released from a ruptured schizont that were stained with Giemsa (d). (e) Fluorescence showing reaction of rabbit-antibodies to the 26 kDa protein. (f) Fluorescence showing the presence of some antigen (arrowed) away from the mature intra-cellular parasite.

with other monoclonal antibodies [26]. Mab 1Cll53 failed to react with parasite antigen in Western blots, suggesting that the epitope recognised by the Mab was not a linear or contiguous epitope but was dependent on the conformation of the protein. The rabbit polyclonal antibodies raised against the purified 26 kDa protein, however, reacted in Western blots with the 26 kDa antigen from parasite material (Fig. 3) and with the affinity purified antigen (data not shown). No

Fig. 2. Immunoprecipitation of [35S]methionine-labelled parasite proteins. Fluorograph of immune-precipitates with (a) control Mab, (b) Mab 1Cll-53. The migration positions of molecular weight markers in kDa and the 26 kDa antigen are arrowed.

130 Relative Fluorescence

(--)

,/.---

-'~200

_. . . . . . . .

-9O

80

~z97

o10

T

"~68 -~43

7o

.~m <

0.05

o ~

i 50

o

-~26

Fig. 3. Autoradiograph of a Western blot of parasite proteins probed with the rabbit polyclonal antibodies raised against the 26 kDa protein. (a) Schizonts, (b) trophozoites, (c) mid-rings. The migration positions of molecular weight standards in kDa are indicated by arrows. (Fig. 4). Nineteen residues were clearly identified by gas phase sequencing of 200 pmol of the purified antigen (Table II). Discussion

The data suggest that the antigen recognised by Mab 1Cll-53 is largely associated with the mature parasite within the red cell. T h e r e were no detectable quantities of the antigen in ring stages. It is possible that the detection of antigen in patches in the red cell cytoplasm is due to the export of some of the antigen into the parasitophorous vacuole, and subsequent transfer to vesicles and M a u r e r ' s clefts. During the rupture of schizonts, the immunofluorescence data suggest that the 26 k D a antigen is released into the medium together with merozoites and this is supported by the detection of the antigen, by Western blotting, in the supernatants of cultures undergoing re-in-

10

20

30

40

50

60

70

80

90

100

110

120

Retention Time (rain)

Fig. 4. Recovery of the 26 kDa antigen from electroeluates by inverse gradient HPLC. Approximately 1 nmol of the antigen purified by SDS-PAGE and electroelution was injected onto an ODS-Hypersil column (0.21x10 cm, 5 ixm particle size) equilibrated with 90% n-propanol/10% water (Solvent A) at a flow rate of 400 Dxlmin-~. A steep linear gradient (0-100% B in 0.1 min) was applied at 50 min and 1.8 min later the flow rate was reduced to 40 ~1 min -1. Solvent B was 50% n-propanol/50% water containing 0.4% (v/v) trifluoroacetic acid. The calculated dead volume of the instrument (Perkin Elmer LC4) between the mixer and the top of the column was 800 ~1. vasion (unpublished data). The in vitro growth inhibition studies showed that Mab 1Cll-53 produced a significant but weak inhibition of parasite growth. The inhibition assay utilised in the present experiments does not distinguish between inhibition of parasite maturation and inhibition of reinvasion. However, it is possible that Mab 1Cll-53 interferes with reinvasion when it binds to antigen released from rupturing schizonts. The degree of inhibition observed is less than the 100% inhibition at 40 and 250 txg m1-1 seen with two Mabs directed against the 80, 66 and 42 k D a rhoptry antigen complex using an identical assay system [4]. Also, using < 2 txg ml i of Mabs, > 7 5 % inhibition of parasite growth has been reported with Mabs against a 195 k D a merozoite surface antigen [2,3]. It would appear therefore that different antigens give rise to antibodies with a wide range of inhibitory capacities. I g G from a polyclonal rabbit antiserum to the 26 k D a protein, unlike the Mab, did not detectably inhibit in the assay, and this parallels the observations made with antibodies to the 220 kDa

131 TABLE II Sequence analysis of the 1Cll-53 antigen. Sequence: Pro-Ile-Pro-Asn-Asn-Pro-Gly-Ala-Gly-Glu-Asn-Ala-Phe-Asp-Pro-Val-PheVal-LysEdman cycle Yield of PTH-derivativesa 1 Pro (46) 2 lie (67) 3 Pro (41) 4 Asn (39) 5 Asn (49) 6 Pro (42) 7 Gly (48) 8 Ala (50) 9 Gly (32) 10 Glu (41) aValues in parentheses are given in pmol. S-antigen of P. falciparum [27]. It is possible that the concentration of specific antibodies against critical epitopes in the 26 kDa antigen in the rabbit IgG preparation is insufficient to produce detectable inhibition. The 26 kDa antigen, like the S-antigen [27], is an example of a parasite antigen that is not exclusively located in either the surface membrane or the rhoptries of merozoites, but is the target of an inhibitory Mab. The weak inhibition suggests that the antigen is not a primary vaccine candidate. However, its role in protective immunity may be difficult to assess by growth inhibition assays alone. The epitope recognised by Mab 1Cll53 is conserved in a number of African and Asian strains of P. falciparum and this is a useful property if an antigen is being considered for use as a component of a malaria vaccine. The lack of labelling of the 26 kDa antigen with [3H]glucosamine and [3H]myristic acid suggests that the antigen is not covalently modified by acylation or glycosylation and this is consistent with a predominantly non-membrane location for the antigen. The absence of a methionine residue at the N-terminus of the protein shows that it has undergone processing after synthesis as is common with eukaryotic proteins. The sequence contains an unusually large (4 out of 19 residues sequenced) proportion of proline, suggesting that this region of the protein is not part of an a-helix. Comparison with published sequences of other P. falciparum proteins, that have been mostly sequenced at the level of D N A rather than protein, confirmed that the sequence obtained was a unique one. It is interesting to note that the amino

Edman cycle 11 12 13 14 15 16 17 18 19

Yield of PTH-derivatives Asn (28) Ala (28) Phe (46) Asp (17) Pro (6) Val (10) Phe (7) Val (60) Lys (10)

terminus of the 26 kDa antigen shows a weak sequence homology to the circumsporozoite protein of P. falciparurn [28], being identical in 7 out of 19 positions with residues 300 to 318 of the circumsporozoite protein. Nineteen residues represents about 8-9% of the amino acids present in a 26 kDa protein. The N-terminal sequence obtained may therefore be sufficient to construct a peptide that can be used for immunisation studies. Parts of the sequence may also be used for constructing oligonucleotide probes for screening c D N A and genomic libraries to isolate clones of the antigen. This may enable the complete structure, and thereby perhaps the function of the protein, to be determined and permit more detailed immunogenicity and protection studies to be performed with either a part or whole of the antigen.

Acknowledgements The technical assistance of R. Lord and W. Relf with parts of the work, the supply of blood from the Red Cross Blood Bank, Brisbane, and parasite material from the Commonwealth Serum Laboratories, Melbourne is greatly appreciated. The authors are grateful to D. Battistutta for help with statistics. This work was supported by the Australian Joint Venture for the Development of a Malaria Vaccine, the National Health and Medical Research Council of Australia, the National Biotechnology Research Grants Scheme and the U N D P / W H O / W o r l d Bank Special Program for Research and Training in Tropical Diseases.

132

References 1 Reese, R.T., Ardeshir, F., Flint, J.E., Howard, R.F., Ramasamy, R., Richman, S.J. and Stanley, H.A. (1985) Use of biotechnology to study Plasmodium falciparum asexual blood stage antigens. In: Proceedings of the Asia and Pacific Conference on Malaria: Practical Considerations on Malaria Vaccines and Clinical Trials (Siddiqui, W.A., ed.), pp. 309-325, University of Hawaii, Honolulu. 2 Schmidt-Ullrich, R., Brown, J., Whittle, H. and Sun-Lin, P. (1986) Human-human hybridomas secreting monoclonal antibodies to the Mr 195 000 Plasmodiurn falciparum blood stage antigen. J. Exp. Med. 163, 179-188. 3 Brown, J., Whittle, H., Berzins, K., Howard, R.J., Marsh, K. and Sjoberg, K. (1986) Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus. Clin. Exp. Immunol. 63, 135-140. 4 Schofield, L., Bushell, G.R., Cooper, J.A., Saul, A.J., Upcroft, J.A. and Kidson, C. (1986) A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognised by inhibitory monoclonal antibodies. Mol. Biochem. Parasitol. 18, 183-195. 5 Hall, R., Hyde, J.E., Goman, M., Simmons, D.L., Hope, I.A., Mackay, M. and Scaife, J. (1984) Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria. Nature 311,379-385. 6 Perrin, L.H., Merkli, B., Chizzolini, C., Loche, M,, Smart, J. and Richle, R. (1984) Anti-malarial immunity in Saimiri monkeys: immunisation with surface components of asexual blood stages. J. Exp. Med. 160, 441-451. 7 Aikawa, M., Miller, L.H., Johnson, J. and Rabbege, J. (1978) Erythrocyte entry by malarial parasites: A moving junction between erythrocyte and parasite. J. Cell. Biol. 77, 72-82. 8 Chen, P., Lamont, G., Elliot, T., Kidson, C., Bronon, G., Mitchell, G., Stace, J. and Alpers, M. (1980) Plasmodium falciparum strains from Papua New Guinea; culture characteristics and drug sensitivity. SE Asian J. Trop. Med. Public Health 11,435-440. 9 Trager, W. and Jensen, J.B. (1976) Human malarial parasites in continuous culture. Science 193, 673-675. 10 Lambros, C. and Vanderberg, J.P. (1979) Synchronisation of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65,418-420. 11 Jensen, J.B. (1978) Concentration from continuous culture of erythrocytes infected with trophozoites and scliizonts of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 27, 1274-1276. 12 Howard, R.F. and Reese, R.T. (1984) Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum. Mol. Biochem. Parasitol. 10, 319-334. 13 Ramasamy, R. and Reese, R.T. (1986) Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins. Mol. Biocliem. Parasitol. 19, 91-101. 14 Haldar, K., Ferguson, M.A.J. and Cross, G.A.M. (1985) Acylation of a Plasmodium falciparum merozoite surface antigen via sn-l,2-diacyl glycerol. J. Biol. Chem. 260, 4969-4974.

15 Ramasamy, R. and Reese, R.T. (1985) A role for carbohydrate moieties in the immune response to malaria. J. Immunol. 134, 1952-1955. 16 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-685. 17 Schneider, C., Newman, R.A., Sutherland, D.R., Asser, U. and Greaves, M.F. (1982) A one-step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem. 257, 10766-10769. 18 Hunkapiller, M.W., Lujan, E., Ostrander, F. and Hood, L.E. (1983) Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 91,227-236. 19 Simpson, R.J., Moritz, R.L., Nice, E.C. and Grego, B. (In Press) A high-performance liquid chromatography procedure for recovering sub-nanomole amounts of protein from SDS-gel electroeluates for gas-phase sequence analysis. Eur. J. Biochem+ 20 Simpson, R.J., Smith, J.A., Moritz, R.L., O'Hare, M.J., Rudland, P.S., Morrison, J.R., Lloyd, C.J., Grego, B.+ Burgess, A.W. and Nice, E.C. (1985) Rat epidermal growth factor: complete amino acid sequence. Eur. J. Biochem. 153,629-637. 21 Klapper, D.G., Wilde, C.E. and Capra, J.D. (1978) Automated amino acid sequence of small peptides using polybrene. Anal. Biochem. 85, 126-131. 22 Heukeshoven, J. and Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112. 23 McBride, J.S., Walliker, D. and Morgan, G. (1982) Antigenic diversity in the human malaria parasite Plasmodiurn falciparum. Science 217,254--257. 24 Myler, P., Saul, A. and Kidson, C. (1983) The synthesis and fate of stage-specific proteins in Plasmodium falciparum cultures. Mol. Biochem. Parasitol. 9, 37-45. 25 Ramasamy, R. (1987) Studies on glycoproteins in the human malaria parasite Plasmodium falciparum-lectin binding properties and the possible carbohydrate-protein linkage. Immun. Cell. Biol. 65, 147-152. 26 Ramasamy, R. (1987) Studies on glycoproteins in the human malaria parasite Plasmodium falciparum. The identification of a myristilated 45 kDa merozoite membrane glycoprotein. Immun. Cell. Biol. 65, 419-424. 27 Saul, A., Myler, P., Schofield, L. and Kidson, C. (1984) A high molecular weight antigen in Plasmodium falciparum recognised by inhibitory monoclonal antibodies. Parasite Immunol, 6, 39-50. 28 Dame, J.B., Williams, J.L., McCutchan, T.F., Weber, J.L., Wirtz, R.A., Hockmeyer, W.T., Maloy, W.L., Haynes, J.D., Schneider, I., Roberts, D., Sanders, G.S., Reddy, E.P., Diggs, C.L. and Miller, L.H. (1984) Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 225,593-599.