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A solid-phase antibody binding-inhibition test, for the assay of Plasmodium berghei antigen and antibodies, using radioiodinated protein A
Journal of Immunological Methods, 32 (1980) 151--155 © Elsevier/North-Holland Biomedical Press
151
A SOLID-PHASE A N T I B O D Y B I N D I N G - I N H I B I T I O N TEST, F O R THE ASSAY OF PLASMODIUM BERGHEI ANTIGEN AND ANTIBODIES, USING RADIOIODINATED PROTEIN A I
HAVA AVRAHAM, DAN T. SPIRA, YORAM GORSKY and DOV SULITZEANU Lautenberg Center for General and Tumor Immunology, and Department of Protozoology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 21 August 1979, accepted 20 September 1979)
Sonicated red blood cells (RBC) of rats infected with Plasmodium berghei (Pb) were used to coat plastic tubes with Pb antigens. The antigen-coated tubes were employed to detect Pb antigens and antibodies, with high efficiency. Anti-Pb antibodies were estimated by treating the tubes with rabbit or rat anti-Pb sera and assaying the bound Ig with radiolabeled Staphylococcus PrA. Pb antigens were detected by their capacity to inhibit the binding of the anti-Pb antibodies. Using a rabbit-Pb serum, sonicated, infected RBC (50% parasitemia) gave detectable inhibition up to 1 : 106 dilution. INTRODUCTION T h e need for an a c c u r a t e and reliable test to d e t e c t low grade malaria i n f e c t i o n s in m a n has been felt for m a n y years. In the last d e c a d e m a n y ' R e s e a r c h r e c o m m e n d a t i o n s ' b y i n t e r n a t i o n a l or regional bodies have design a t e d i m m u n o d i a g n o s i s as one o f the targets f o r f u t u r e malaria research (W.H.O. T e c h n . Rep. Ser. 1 9 6 8 , 1 9 7 4 , 1975). T h e 'Special P r o g r a m f o r R e s e a r c h and Training in T r o p i c a l Diseases' again listed it as one o f the preferred fields (W.H.O., 1976). Yet, screening o f the literature reveals that, so far, o n l y a t t e m p t s t o d e t e c t a n t i b o d y have been p u r s u e d , either b y classical serological m e t h o d s or b y the m o r e sensitive r a d i o i m m u n o a s s a y or E L I S A methods. In this c o m m u n i c a t i o n we describe a r a d i o i m m u n o a s s a y , p o t e n t i a l l y capable n o t o n l y to m e a s u r e a n t i b o d y b u t also t o d e t e c t m i n u t e quantities o f malarial antigen in the b l o o d . MATERIALS AND METHODS Animals O u t b r e d , locally g r o w n rats, were i n f e c t e d with P l a s m o d i u m berghei (Pb) as described b e f o r e (Spira et al., 1 9 7 6 ) . I This work comprises part of the Ph.D. thesis of H. Avraham.
152
Buffers Unless otherwise stated, all dilutions were made in borate-buffered saline (BBS), pH 8. Tubes were washed with BBS containing 0.1% Tween 20. Reticulocy tes Rats were injected with 8 m g/ 100 g phenyl hydrazi ne (Zuckerman and Spira, 1961). Blood was collected 3--4 days later, at which time 60--70% of the red cells were reticulocytes. Pb antigens Two antigen preparations were used: (a) Pb extract, prepared by treating Pb-infected rat RBC with saponin (Zuckerman et al., 1967); (b) sonicated, infected RBC (SIRC): blood was collected from Pb-infected rats at the height of parasitemia in tubes containing heparin. The RBC were washed 3 times, then resuspended in 10 vol BBS and sonicated at 100 W for 1 min in an AMC ultrasonic vibrator. The sonicate was centrifuged to remove insoluble material and was used as antigen. Identical preparations from uninfected rat RBC served as controls. Radioiodinated Protein A Protein A (PrA) (Pharmacia, Uppsala, Sweden) was labeled with ~25I ([12sI]PrA) as described by Zeltzer and Seeger (1977). The a m o u n t used per test was 50,000 counts/min, corresponding to a p p r o x i m a t e l y 10 ng protein. The labeled material was diluted in 10% fetal calf serum (FCS) in BBS. Anti-Pb sera Rabbit anti-Pb serum was prepared by injecting into rabbits Pb extract in c o mp lete Freund's adjuvant (Spira and Zuckerman, 1962). Serum was collected after one or several injection courses. Both the antiserum and the normal rabbit serum c o n t r o l (NRS) were absorbed with normal rat red blood cells (RBC), with normal rat reticulocytes and with glutaraldehyde-polymerized rat serum (Avrameas and T e r n y n c k , 1969), to remove antibodies to rat antigens. To prepare rat antisera, the animals were given 3 injections of parasitized red blood cells (106 parasites per injection) at fortnightly intervals. Two weeks after the last injection the rats were bled and the sera were pooled. Antisera were divided into small aliquots and stored at --20 ° C. Assay technique Biological reagents were centrifuged for 0.5 h at 12,000 × g before use, p o l y s t y r e n e tubes (100 mm × 13 mm, Nunc, Denmark) were treated with 0.1% glutaraldehyde, as instructed by Barrett (1977). To each tube, 0.5 ml SIRC was added. The tubes were kept for 15 h at 4°C and were then washed 3 times and treated overnight with 0.7 ml 0.01% bovine serum albumin in phosphate-buffered saline. To assay for the binding of antibodies, 0.4 ml volumes of the antiserum, diluted in 10% FCS as required, were added to the
153 a n t i g e n - c o a t e d t u b e s a n d the t u b e s were r o t a t e d o v e r n i g h t at 4°C in a tissue culture r o t a t o r . A f t e r washing 3 times, 0.4 ml labeled Protein A were a d d e d and the t u b e s were r o t a t e d for 0.5 h at 4°C. The r e a c t i o n was s t o p p e d b y diluting the Protein A with 4 ml BBS, r a d i o a c t i v i t y in the t u b e s was measured, the t u b e s were w a s h e d and c o u n t e d again to d e t e r m i n e the b o u n d r a d i o a c t i v i t y . F o r the inhibition tests, various dilutions o f the i n h i b i t o r were m i x e d with an equal v o l u m e o f the a p p r o p r i a t e l y diluted a n t i s e r u m and the m i x t u r e was i n c u b a t e d for 0.5 h at 37°C, f o l l o w e d b y 24 h at 4°C. The residual binding activity was t h e n m e a s u r e d as before. All tests were d o n e in duplicate. T o m i n i m i z e variability a t t r i b u t a b l e to changes in sonicate c o n c e n t r a t i o n , S I R C dilutions were m a d e in sonicate o f n o r m a l rat red cells. RESULTS
Binding of anti-Pb antibodies to tubes coated with SIRC C o a t e d t u b e s were t r e a t e d with various dilutions o f r a b b i t anti-Pb serum. Binding titer was at least 1 : 3 1 2 5 (Table 1). N R S gave negligible binding. The rat a n t i s e r u m (Table 2) h a d an even higher binding titer (over 1 : 1 5 . 6 2 5 ) , b u t the binding o f n o r m a l rat serum was also higher. All sera gave negligible binding w h e n r e a c t e d with u n c o a t e d tubes or with t u b e s c o a t e d with sonicate o f u n i n f e c t e d rat red cells (data n o t s h o w n ) .
Inhibition o f binding of anti-Pb antibody by SIRC These e x p e r i m e n t s were carried o u t b o t h to c h e c k the specificity o f the binding and to d e t e r m i n e the sensitivity o f the t e c h n i q u e f o r the. d e t e c t i o n o f the plasmodial antigen. R a b b i t or rat antisera (diluted 1 : 2 5 0 ) were i n c u b a t e d with 10-fold dilutions o f the S I R C , after w h i c h the residual bind-
TABLE 1 BINDING OF RABBIT ANTI-Pb SERUM TO TUBES COATED WITH SIRC Tubes coated with SIRC were treated with various dilutions of NRS or of rabbit anti-Pb serum, washed, then treated with [125I]PrA. The net binding is calculated as the difference b e t w e e n rabbit anti-Pb serum and NRS. Serum
Per cent binding
N e t binding (%)
dilution
1 1 1 1 1
: 25 : 125 : 625 : 3125 : 15,625
NRS
Rabbit anti-Pb serum
0.5 0.1 0.1 0.1 0.1
27.1 23.4 10.2 2.8 0.5
26.6 23.0 10.1 2.7 0.5
154 TABLE 2 BINDING RAT ANTI-Pb SERUM TO TUBES COATED WITH SIRC Procedure as in Table 1. Serum dilution
1 1 1 1 1 1 1
Per cent binding
: 25 : 125 : 625 : 3125 : 15,625 : 78,125 : 390,625
Net binding (%)
Normal rat serum
Rat antiPb serum
5.3 5.5 5.0 4.5 4.0 4.0 3.8
20.1 18.4 15.8 8,7 7.7 5.6 5.3
14.8 12.9 10.8 4.2 3.7 1.6 1.5
i n g a c t i v i t y w a s a s s a y e d . As s e e n i n Fig. 1, S I R C i n h i b i t e d p r a c t i c a l l y c o m pletely the b i n d i n g of b o t h antisera, t h u s a t t e s t i n g to the specificity of the r e a c t i o n . However, the i n h i b i t i o n titer was m u c h higher with the r a b b i t a n t i s e r u m , w h i c h could detect SIRC at 1 : 1 0 6 dilution, corresponding t h e o r e t i c a l l y to a p a r a s i t e m i a of 0 . 0 0 0 0 5 % . These results were c o n f i r m e d in 8 r e p e a t e x p e r i m e n t s . S o n i c a t e s o f n o n - i n f e c t e d r e d b l o o d cells o r o f r e t i c u l o c y t e s gave n o i n h i b i t i o n w h a t s o e v e r .
IIIHIBITIOH OF 81NOIMG OF AHTI-Pb ANTIBOOIE$ BY Pb ARTIGEH 100 8O
R•e• I I
RABBIT \anti-Pb
'Z o:: 20 0
RA
~anti-Pb
•
N
Iserum e~ 1 .!10 1:10 ' z 1:103 ~ . u ~1:10 . u , ~~'.1:10 - - . .s- L1:106 - - ~ e -'i:107 ~,NTIGEN DILUTIONS
Fig. 1. Inhibition of binding of Pb antibodies by SIRC. Rabbit or rat antisera, diluted 1 : 250, were incubated with various dilutions of SIRC, after which the residual binding activity was assayed. Results are expressed as per cent inhibition.
155 DISCUSSION T h e a n t i b o d y b i n d i n g - i n h i b i t i o n t e c h n i q u e d e v e l o p e d in this w o r k a p p e a r s to be o f p r o m i s e f o r assaying b o t h a n t i m a l a r i a l a n t i b o d i e s a n d malarial antigens. T h e t e c h n i q u e is highly sensitive; a n t i b o d i e s were still d e t e c t a b l e in s e r u m diluted 1 / 1 5 , 0 0 0 , while antigen was d e t e c t a b l e at a c o n c e n t r a t i o n r o u g h l y e q u i v a l e n t to t h a t f o u n d in b l o o d w i t h 5 X 1 0 - s % p a r a s i t e m i a . It a p p e a r s likely, t h e r e f o r e , t h a t a t e c h n i q u e based on a similar a p p r o a c h m a y e v e n t u a l l y lead to an i m m u n o d i a g n o s t i c t e s t f o r m a l a r i a in m a n , based on t h e i d e n t i f i c a t i o n o f t h e parasites in the b l o o d . O f p a r t i c u l a r i m p o r t a n c e f r o m the p r a c t i c a l p o i n t o f view was t h e finding t h a t s i m p l e s o n i c a t i o n o f t h e i n f e c t e d b l o o d y i e l d e d a p r e p a r a t i o n w h i c h was highly e f f e c t i v e in t h e i n h i b i t i o n assays. F u r t h e r e x p e r i m e n t s are u n d e r w a y to ascertain w h e t h e r a similar t e s t can be d e v e l o p e d f o r the d e t e c t i o n o f h u m a n malaria. ACKNOWLEDGEMENTS This s t u d y has b e e n s u p p o r t e d b y research grants f r o m t h e L a u t e n b e r g E n d o w m e n t F u n d a n d b y the C e n t e r f o r S t u d y o f T r o p i c a l a n d I n f e c t i o u s Diseases o f the H e b r e w University. REFERENCES Avrameas, S. and T. Ternynck, 1969, Immunochemistry 6, 53. Barrett, M.J., 1977, US Patent No. 4.001.583. Spira, D.T. and A. Zuckerman, 1962, Science 137,536. Spira, D.T., J. Golenser and I. Gery, 1976, Clin. Exp. Immunol. 23, 139. W.H.O. Techn. Rep. Ser., 1968, Immunology of Malaria (W.H.O., Geneva) p. 396. W.H.O. Techn. Rep. Ser., 1974, 543. W.H.O. Techn. Rep. Ser., 1975, Developments in Malaria Immunology (W.H.O., Geneva) p. 597. W.H.O./TDR/IM, 1976, Report of the Scientific Working Group on the Immunology of Malaria (W.H.O., Geneva) p. 76.3. Zeltzer, P.M. and R.C. Seeger, 1977, J. Immunol. Methods 17, 163. Zuckerman, A. and D.T. Spira, 1961, J. Infect. Dis. 108, 339. Zuckerman, A., D.T. Spira and Y. Hamburger, 1967, Bull. W.H.O. 37,431.
151
A SOLID-PHASE A N T I B O D Y B I N D I N G - I N H I B I T I O N TEST, F O R THE ASSAY OF PLASMODIUM BERGHEI ANTIGEN AND ANTIBODIES, USING RADIOIODINATED PROTEIN A I
HAVA AVRAHAM, DAN T. SPIRA, YORAM GORSKY and DOV SULITZEANU Lautenberg Center for General and Tumor Immunology, and Department of Protozoology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 21 August 1979, accepted 20 September 1979)
Sonicated red blood cells (RBC) of rats infected with Plasmodium berghei (Pb) were used to coat plastic tubes with Pb antigens. The antigen-coated tubes were employed to detect Pb antigens and antibodies, with high efficiency. Anti-Pb antibodies were estimated by treating the tubes with rabbit or rat anti-Pb sera and assaying the bound Ig with radiolabeled Staphylococcus PrA. Pb antigens were detected by their capacity to inhibit the binding of the anti-Pb antibodies. Using a rabbit-Pb serum, sonicated, infected RBC (50% parasitemia) gave detectable inhibition up to 1 : 106 dilution. INTRODUCTION T h e need for an a c c u r a t e and reliable test to d e t e c t low grade malaria i n f e c t i o n s in m a n has been felt for m a n y years. In the last d e c a d e m a n y ' R e s e a r c h r e c o m m e n d a t i o n s ' b y i n t e r n a t i o n a l or regional bodies have design a t e d i m m u n o d i a g n o s i s as one o f the targets f o r f u t u r e malaria research (W.H.O. T e c h n . Rep. Ser. 1 9 6 8 , 1 9 7 4 , 1975). T h e 'Special P r o g r a m f o r R e s e a r c h and Training in T r o p i c a l Diseases' again listed it as one o f the preferred fields (W.H.O., 1976). Yet, screening o f the literature reveals that, so far, o n l y a t t e m p t s t o d e t e c t a n t i b o d y have been p u r s u e d , either b y classical serological m e t h o d s or b y the m o r e sensitive r a d i o i m m u n o a s s a y or E L I S A methods. In this c o m m u n i c a t i o n we describe a r a d i o i m m u n o a s s a y , p o t e n t i a l l y capable n o t o n l y to m e a s u r e a n t i b o d y b u t also t o d e t e c t m i n u t e quantities o f malarial antigen in the b l o o d . MATERIALS AND METHODS Animals O u t b r e d , locally g r o w n rats, were i n f e c t e d with P l a s m o d i u m berghei (Pb) as described b e f o r e (Spira et al., 1 9 7 6 ) . I This work comprises part of the Ph.D. thesis of H. Avraham.
152
Buffers Unless otherwise stated, all dilutions were made in borate-buffered saline (BBS), pH 8. Tubes were washed with BBS containing 0.1% Tween 20. Reticulocy tes Rats were injected with 8 m g/ 100 g phenyl hydrazi ne (Zuckerman and Spira, 1961). Blood was collected 3--4 days later, at which time 60--70% of the red cells were reticulocytes. Pb antigens Two antigen preparations were used: (a) Pb extract, prepared by treating Pb-infected rat RBC with saponin (Zuckerman et al., 1967); (b) sonicated, infected RBC (SIRC): blood was collected from Pb-infected rats at the height of parasitemia in tubes containing heparin. The RBC were washed 3 times, then resuspended in 10 vol BBS and sonicated at 100 W for 1 min in an AMC ultrasonic vibrator. The sonicate was centrifuged to remove insoluble material and was used as antigen. Identical preparations from uninfected rat RBC served as controls. Radioiodinated Protein A Protein A (PrA) (Pharmacia, Uppsala, Sweden) was labeled with ~25I ([12sI]PrA) as described by Zeltzer and Seeger (1977). The a m o u n t used per test was 50,000 counts/min, corresponding to a p p r o x i m a t e l y 10 ng protein. The labeled material was diluted in 10% fetal calf serum (FCS) in BBS. Anti-Pb sera Rabbit anti-Pb serum was prepared by injecting into rabbits Pb extract in c o mp lete Freund's adjuvant (Spira and Zuckerman, 1962). Serum was collected after one or several injection courses. Both the antiserum and the normal rabbit serum c o n t r o l (NRS) were absorbed with normal rat red blood cells (RBC), with normal rat reticulocytes and with glutaraldehyde-polymerized rat serum (Avrameas and T e r n y n c k , 1969), to remove antibodies to rat antigens. To prepare rat antisera, the animals were given 3 injections of parasitized red blood cells (106 parasites per injection) at fortnightly intervals. Two weeks after the last injection the rats were bled and the sera were pooled. Antisera were divided into small aliquots and stored at --20 ° C. Assay technique Biological reagents were centrifuged for 0.5 h at 12,000 × g before use, p o l y s t y r e n e tubes (100 mm × 13 mm, Nunc, Denmark) were treated with 0.1% glutaraldehyde, as instructed by Barrett (1977). To each tube, 0.5 ml SIRC was added. The tubes were kept for 15 h at 4°C and were then washed 3 times and treated overnight with 0.7 ml 0.01% bovine serum albumin in phosphate-buffered saline. To assay for the binding of antibodies, 0.4 ml volumes of the antiserum, diluted in 10% FCS as required, were added to the
153 a n t i g e n - c o a t e d t u b e s a n d the t u b e s were r o t a t e d o v e r n i g h t at 4°C in a tissue culture r o t a t o r . A f t e r washing 3 times, 0.4 ml labeled Protein A were a d d e d and the t u b e s were r o t a t e d for 0.5 h at 4°C. The r e a c t i o n was s t o p p e d b y diluting the Protein A with 4 ml BBS, r a d i o a c t i v i t y in the t u b e s was measured, the t u b e s were w a s h e d and c o u n t e d again to d e t e r m i n e the b o u n d r a d i o a c t i v i t y . F o r the inhibition tests, various dilutions o f the i n h i b i t o r were m i x e d with an equal v o l u m e o f the a p p r o p r i a t e l y diluted a n t i s e r u m and the m i x t u r e was i n c u b a t e d for 0.5 h at 37°C, f o l l o w e d b y 24 h at 4°C. The residual binding activity was t h e n m e a s u r e d as before. All tests were d o n e in duplicate. T o m i n i m i z e variability a t t r i b u t a b l e to changes in sonicate c o n c e n t r a t i o n , S I R C dilutions were m a d e in sonicate o f n o r m a l rat red cells. RESULTS
Binding of anti-Pb antibodies to tubes coated with SIRC C o a t e d t u b e s were t r e a t e d with various dilutions o f r a b b i t anti-Pb serum. Binding titer was at least 1 : 3 1 2 5 (Table 1). N R S gave negligible binding. The rat a n t i s e r u m (Table 2) h a d an even higher binding titer (over 1 : 1 5 . 6 2 5 ) , b u t the binding o f n o r m a l rat serum was also higher. All sera gave negligible binding w h e n r e a c t e d with u n c o a t e d tubes or with t u b e s c o a t e d with sonicate o f u n i n f e c t e d rat red cells (data n o t s h o w n ) .
Inhibition o f binding of anti-Pb antibody by SIRC These e x p e r i m e n t s were carried o u t b o t h to c h e c k the specificity o f the binding and to d e t e r m i n e the sensitivity o f the t e c h n i q u e f o r the. d e t e c t i o n o f the plasmodial antigen. R a b b i t or rat antisera (diluted 1 : 2 5 0 ) were i n c u b a t e d with 10-fold dilutions o f the S I R C , after w h i c h the residual bind-
TABLE 1 BINDING OF RABBIT ANTI-Pb SERUM TO TUBES COATED WITH SIRC Tubes coated with SIRC were treated with various dilutions of NRS or of rabbit anti-Pb serum, washed, then treated with [125I]PrA. The net binding is calculated as the difference b e t w e e n rabbit anti-Pb serum and NRS. Serum
Per cent binding
N e t binding (%)
dilution
1 1 1 1 1
: 25 : 125 : 625 : 3125 : 15,625
NRS
Rabbit anti-Pb serum
0.5 0.1 0.1 0.1 0.1
27.1 23.4 10.2 2.8 0.5
26.6 23.0 10.1 2.7 0.5
154 TABLE 2 BINDING RAT ANTI-Pb SERUM TO TUBES COATED WITH SIRC Procedure as in Table 1. Serum dilution
1 1 1 1 1 1 1
Per cent binding
: 25 : 125 : 625 : 3125 : 15,625 : 78,125 : 390,625
Net binding (%)
Normal rat serum
Rat antiPb serum
5.3 5.5 5.0 4.5 4.0 4.0 3.8
20.1 18.4 15.8 8,7 7.7 5.6 5.3
14.8 12.9 10.8 4.2 3.7 1.6 1.5
i n g a c t i v i t y w a s a s s a y e d . As s e e n i n Fig. 1, S I R C i n h i b i t e d p r a c t i c a l l y c o m pletely the b i n d i n g of b o t h antisera, t h u s a t t e s t i n g to the specificity of the r e a c t i o n . However, the i n h i b i t i o n titer was m u c h higher with the r a b b i t a n t i s e r u m , w h i c h could detect SIRC at 1 : 1 0 6 dilution, corresponding t h e o r e t i c a l l y to a p a r a s i t e m i a of 0 . 0 0 0 0 5 % . These results were c o n f i r m e d in 8 r e p e a t e x p e r i m e n t s . S o n i c a t e s o f n o n - i n f e c t e d r e d b l o o d cells o r o f r e t i c u l o c y t e s gave n o i n h i b i t i o n w h a t s o e v e r .
IIIHIBITIOH OF 81NOIMG OF AHTI-Pb ANTIBOOIE$ BY Pb ARTIGEH 100 8O
R•e• I I
RABBIT \anti-Pb
'Z o:: 20 0
RA
~anti-Pb
•
N
Iserum e~ 1 .!10 1:10 ' z 1:103 ~ . u ~1:10 . u , ~~'.1:10 - - . .s- L1:106 - - ~ e -'i:107 ~,NTIGEN DILUTIONS
Fig. 1. Inhibition of binding of Pb antibodies by SIRC. Rabbit or rat antisera, diluted 1 : 250, were incubated with various dilutions of SIRC, after which the residual binding activity was assayed. Results are expressed as per cent inhibition.
155 DISCUSSION T h e a n t i b o d y b i n d i n g - i n h i b i t i o n t e c h n i q u e d e v e l o p e d in this w o r k a p p e a r s to be o f p r o m i s e f o r assaying b o t h a n t i m a l a r i a l a n t i b o d i e s a n d malarial antigens. T h e t e c h n i q u e is highly sensitive; a n t i b o d i e s were still d e t e c t a b l e in s e r u m diluted 1 / 1 5 , 0 0 0 , while antigen was d e t e c t a b l e at a c o n c e n t r a t i o n r o u g h l y e q u i v a l e n t to t h a t f o u n d in b l o o d w i t h 5 X 1 0 - s % p a r a s i t e m i a . It a p p e a r s likely, t h e r e f o r e , t h a t a t e c h n i q u e based on a similar a p p r o a c h m a y e v e n t u a l l y lead to an i m m u n o d i a g n o s t i c t e s t f o r m a l a r i a in m a n , based on t h e i d e n t i f i c a t i o n o f t h e parasites in the b l o o d . O f p a r t i c u l a r i m p o r t a n c e f r o m the p r a c t i c a l p o i n t o f view was t h e finding t h a t s i m p l e s o n i c a t i o n o f t h e i n f e c t e d b l o o d y i e l d e d a p r e p a r a t i o n w h i c h was highly e f f e c t i v e in t h e i n h i b i t i o n assays. F u r t h e r e x p e r i m e n t s are u n d e r w a y to ascertain w h e t h e r a similar t e s t can be d e v e l o p e d f o r the d e t e c t i o n o f h u m a n malaria. ACKNOWLEDGEMENTS This s t u d y has b e e n s u p p o r t e d b y research grants f r o m t h e L a u t e n b e r g E n d o w m e n t F u n d a n d b y the C e n t e r f o r S t u d y o f T r o p i c a l a n d I n f e c t i o u s Diseases o f the H e b r e w University. REFERENCES Avrameas, S. and T. Ternynck, 1969, Immunochemistry 6, 53. Barrett, M.J., 1977, US Patent No. 4.001.583. Spira, D.T. and A. Zuckerman, 1962, Science 137,536. Spira, D.T., J. Golenser and I. Gery, 1976, Clin. Exp. Immunol. 23, 139. W.H.O. Techn. Rep. Ser., 1968, Immunology of Malaria (W.H.O., Geneva) p. 396. W.H.O. Techn. Rep. Ser., 1974, 543. W.H.O. Techn. Rep. Ser., 1975, Developments in Malaria Immunology (W.H.O., Geneva) p. 597. W.H.O./TDR/IM, 1976, Report of the Scientific Working Group on the Immunology of Malaria (W.H.O., Geneva) p. 76.3. Zeltzer, P.M. and R.C. Seeger, 1977, J. Immunol. Methods 17, 163. Zuckerman, A. and D.T. Spira, 1961, J. Infect. Dis. 108, 339. Zuckerman, A., D.T. Spira and Y. Hamburger, 1967, Bull. W.H.O. 37,431.