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Antibodies to diethylcarbamazine cross-react with microfilariae of Wuchereria bancrofti
Immunology Letters, 17 (1988) 7-11 Elsevier IML 00982
Antibodies to diethylcarbamazine cross-react with microfilariae of
Wuchereria bancrofti B. Ravindran, A. K. S a t a p a t h y a n d N. M. P a t t n a i k Regional Medical Research Centre (Indian Council o f Medical Research), Bhubaneswar, lndia (Received 2 September 1987; accepted 8 Scptember 1987)
I. Summary Antibodies directed against the microfilarial sheath have been instrumental in the immune elimination of circulating microfilariae in human lymphatic filariasis. We report here that antibodies to diethyicarbamazine (DEC, the most commonly used anti-filarial drug) cross-react with the sheath of Wuchereria bancrofti microfilariae. Antibodies with reactivity to DEC were raised in rabbits by immunization with a conjugate of methylpiperazine carboxylic acid (MPCA, an acid hydrolysis product of DEC) coupled to bovine serum albumin. The reactivity of these antibodies with microfilarial sheath of W. bancrofti was demonstrated by indirect immunofluorescent assay and indirect immunoperoxidase assay. This reactivity could be effectively inhibited by pre-incubation of the antisera with different haptens such as DEC, MPCA or piperazine citrate.
strable activity against the parasites in vitro [I]. It is generally believed that DEC acts on the microfilariae in conjunction with the host's immune system [2]. We recently produced rabbit antibodies with reactivity to DEC in order to develop an immunoassay for the drug, to use as a tool for studying its mode of action [3]. During the course of further investigations it was found that antibodies to DEC (antiDEC) also reacted with microfilarial sheath of Wuchereria bancrofti. In the present paper we demonstrate the reactivity of anti-DEC towards microfilariae (mf) as shown by indirect immunofluorescent assay (IFA), indirect immunoperoxidase assay (IPA) and enzyme-linked immunoabsorbant assay (ELISA). The reactivity of anti-DEC to microfilarial sheath could be effectively inhibited by pre-incubation of anti-DEC with different haptens such as DEC, methylpiperazine carboxylic acid (MPCA) and piperazine citrate.
3. Materials and Methods 2. Introduction Diethylcarbamazine (DEC) has been in extensive use for the treatment and control of human lymphatic filariasis. Although it has been found to be very effective against the circulating microfilariae under in vivo conditions, the drug has no demonKey words." Filariasis; Wuchereria bancrofti; Diethylcarbamazine; Anti-drug antibody; Surface antigen Correspondence to: Dr. B. Ravindran, Regional Medical Research Centre (I.C.M.R.), P.O. Sainik School, Bhubaneswar 751005, India.
3.1. Animals Male albino rabbits weighing 1.5 to 2 kg were used to raise antisera. 3.2. Chemicals and reagents Diethylcarbamazine citrate and primaquine were kind gifts from Indian Drugs and Detergents, Cuttack, India and Sigma Chemicals Co., USA, respectively. Anti-rabbit immunoglobulins coupled to F1TC or peroxidase were purchased from Dakopatts, Denmark. Tetanus toxin was from Central Research Institute, Kasauli, India.
0165-2478 / 88 / $ 3.50 fc~: 1988 Elsevier Science Publishers B.V. (Biomedical Division)
7
3.3. Conjugate and antisera Detailed methods for the preparation of conjugate and raising antibodies with reactivity to DEC are described by us elsewhere [3]. Briefly, methyl piperazine carboxylic acid (MPCA - an acid hydrolysis product of DEC) was coupled to bovine serum albumin (BSA) or tetanus toxoid (TT) by carbodiimide reaction. The resultant conjugate M P C A - BSA was used to raise antibodies in rabbits and M P C A - T T was used for testing in ELISA. Purified IgG from the immunized sera after absorption with insolubilised BSA was found to react with DEC, as shown by inhibition ELISA [3]. For preparation o f MPCA-liposome, hand-shaken liposomes were prepared [4] with total hen's egg yolk phospholipids which contain about 15% phosphatidylethanolamine. The liposome suspension was mixed with M P C A and EDCI added. Under these conditions all the M P C A coupled will remain on the outermost surface of the multilamellar liposomes. One ml of MPCA-liposomes with or without complete Freund's adjuvant was used per dose for immunization of rabbits. Antisera were also raised using a similar immunization protocol against a conjugate of BSA coupled to primaquine by carbodiimide reaction [3, 5]. These sera served as controls to study the carrier molecule effect, adjuvant effect and the effect of coupling reagent namely 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide (EDCI). 3.4. W. bancrofti microfilariae Ten ml of nocturnal blood were collected in ACD from m f carriers. After removal of plasma the m f were purified by filtration through 5 ~M polycarbonate membranes (Nuclepore Inc., USA) using T r i s - E D T A buffered saline (TEBS: Tris 2 mM, EDTA 2 mM, KCI 0.3 g/l, NaCI 8.0 g/i, glucose 2.0 g/l, pH 7.2). Viable sheathed m f were washed with TEBS and used for preparation of "antigen slides" for IFA and IPA. A somatic antigen extract was also prepared through ultrasonication as described by us previously [61. 3.5. Indirect irnmunofluorescent assay (1FA) Purified m f were spotted on microslides, air-dried and fixed with acetone for 30 min and then stored at - I0 °C until further use. The microslides were examined under the microscope before performing the
assay and only slides containing 10-15 sheathed m f were used. About 15 /zl of serum or its dilutions were applied to the slides and incubated in a humid chamber for 2 h at 37 °C. The slides were then washed thrice with PBS and about 15 ~1 of 100-fold diluted swine anti-rabbit immunoglobulin-FlTC conjugate were added to the spots and incubated at 4 ° C overnight. They were then extensively washed with PBS and mounted in 30°7o glycerol in PBS and examined under a Nikon model Labophot fluorescent microscope. Since reactions to the sheath and to the cuticle are independent, care was taken to differentiate these structures under the fluorescent microscope. 3.6. Indirect immunoperoxidase assay (IPA) Essentially the procedure as described above for IFA was followed with the following differences. The antigen slides were pretreated for 20 min with 0.5°7o H 2 0 2 in methanol to inactivate endogenous peroxidase activity. Swine anti-rabbit immunoglobulin conjugated with peroxidase was used as a second antibody and the reaction was visualized under the light microscope after staining the slides with diaminobenzidine (50 mg/100 ml) in Tris-HCI buffer pH 8.8. 3.7. Inhibition of IFA reactivity with haptens Two hundred tzl of anti-DEC serum (1/4 diluted in PBS with 1°70BSA) were mixed with 200/~1 of various concentrations of DEC or M P C A er piperazine citrate in PBS, pH 7.2, and incubated at 37 °C for 1 h and then spotted on m f 'antigen slides'. The slides were then processed as mentioned above for IFA. The minimum concentration of the hapten that completely inhibits the anti-DEC reactivity to m f sheath was assessed by visual reading under the fluorescent microscope. 3.8. ELISA The detailed procedure is described elsewhere [3]. For coating, sonicated m f antigen was used at a concentration of 9/~g/ml and T T - M P C A at a concentration of 15 #g/ml in PBS. The rabbit sera were titrated and the bound antibody activity was detected by using swine anti-rabbit Ig conjugated to peroxidase (1/500 dilution). The enzyme activity was measured using O-phenylenediamine in citrate phosphate buffer, p H 5.5. After stopping the reaction with 8 N H2SO 4 the OD were measured at
492 nm using a micro-ELISA reader (Bio-Rad, USA). The highest dilution of test serum with three times or more OD as compared to normal rabbit serum was expressed as its titre.
°
°
4. Results
4.1. Reactivity with microfilariae Absorbed anti-DEC serum raised (using B S A - M P C A ) in 5 different rabbits was found to react with m f sheath o f W. bancrofti as shown by I FA and IPA (Table 1). The titre (mf reactivity) of the sera ranged from 1/8 to 1/64. The reaction was restricted only to the m f sheath and not to the cuticle (Fig. 1). Ex-sheathed parasites showed no reactivity with anti-DEC and none of the 5 pre-immunization sera reacted with m f sheath. The anti-DEC sera were tested against each of the preparations of m f collected from 10 different asymptomatic m f carriers and were found to be uniformly reactive. The rabbit (R-719) immunized with MPCA-liposomes in conjunction with CFA also showed anti-DEC activity and reacted with m f sheath (Table 1) while rabbit R-718 in which MPCA-liposomes were administered without any adjuvant failed to elicit detectable levels of anti-DEC during the period of observation. The anti m f titre of the immune sera as shown by Table 1 A n t i b o d y titres in i m m u n i z e d rabbit sera. a Rabbit No.
R-45 R-46 R-305 R-901 R-546 R-718 R-719 R-715 R-716
M a t e r i a l used b
M P C A - BSA M P C A - BSA M P C A - BSA M P C A - BSA M P C A - BSA MPCA - liposomes MPCA - liposomes P Q - BSA P Q - BSA
Batch
1 [ I I II
Anti-DEC titre ¢
Anti-mr titre c
ELtSA
IFA
IPA
6400 12800 12800 25600 25600 50 3200 <50 <50
8 64 16 8 32 NR 4 NR NR
NT 64 8 8 32 NR 4 NR NR
a Sera collected between 3 0 - 4 0 days after i m m u n i z a t i o n were used for the assays. h All the a n i m a l s except R-718 received the respective c o n j u g a t e s mixed with C F A . R e c i p r o c a l of titre. NT not tested; NR = non-reactive.
Fig. 1. Indirect i m m u n o p e r o x i d a s e assay-reactivity o f a n t i - D E C a g a i n s t microfilaria¢. (a) 100 × ; ( b ) 4 0 0 × ; (c) reactivity only to the sheath and not to the cuticle (arrow) (40 ×).
IPA or IFA was, however, very low as compared to their anti-DEC activity as revealed by ELISA (Table 1). Sera collected from two rabbits immunized with PQ-BSA showed no reactivity to m f sheath (Table 1). Affinity purified anti-DEC antibodies were found to react weakly to the surface of sheathed m f as shown by IPA and IFA. Rabbit sera (anti-DEC) collected sequentially during the course of immunization were tested by ELISA against M P C A - T T and the somatic antigen preparation of mf. While the reactivity to M P C A - T T increased very appreciably during the course of immunization, the anti-mr activity increased only 2- to 4-fold (Fig. 2). 4.2. Inhibition of anti-sheath activity The reactivity of anti-DEC to m f sheath could be effectively inhibited by pre-incubation of the sera with various haptens such as DEC, M P C A and piperazine citrate (Table 2). Pre-incubation with normal human sera, however, did not inhibit the reactivity.
Table 2 M i n i m u m c o n c e n t r a t i o n CmM) required for i n h i b i t i o n of antisheath activity ([FA). H a p t e n used
R-46
R-305
R-901
DEC MPCA P i p e r a z i n e citrate
1.25 5
1.25 5 5
1.25 5
:.L-
1.2-
E
}.0-
C'4
"7- -, -
-3'
0.6 i (D
j'
0
/ ./"
,// •. . . . . . . . . .
l~
l
Q~
..
_~×lr•.
G
~0
.:...:...
20
-. : X X T y ~ L
30
-X
--~T
~
<[
DAYS Fig. 2. I':I.ISA-anti-DEC activity of 1RS and their reactivity to somatic antigen of mf. R-546 • - . ; R - 9 0 1 • - - - * ; R - 7 1 5 x - x and R-716 x - - - x tested against M P C A - T T . R-546 • - • and R-901 • --- • tested against T T (sera diluted 100-fold for the above assays). R-546 o - o and R-901 o--- o tested against m f antigen; 1/10 diluled sera were used for testing.
5. Discussion
The present study was undertaken after our initial fortuitous observation that antibodies with reactivity to DEC (anti-DEC) could cross-react with the surface of W. bancrofti m f as shown by IPA and IFA. Detailed investigations with all our anti-DEC sera revealed their uniform reactivity to m f collected from the peripheral blood o f ten different asymptomatic carriers. The reactivity, however, was restricted only to the m f sheath and not to the cuticle. Their titre against m f sheath was very low as compared to their anti-DEC activity, notwithstanding the differences in the sensitivity of ELISA and IFA or IPA. This is further corroborated by the degree of anti10
DEC reactivity against T T - M P C A and sonicated m f antigen as revealed by ELISA (Fig. 2). Sera from rabbits immunized with a conjugate of BSA coupled to primaquine (PQ) failed to react with m f by any of the three different tests. Similarly they showed no reactivity with M P C A - B S A (Fig. 2) although their anti-PQ titre was very high in ELISA (data not shown). These animals had received quantities of BSA and CFA comparable to those of rabbits in which anti-DEC was raised using B S A - M P C A . In both cases the respective haptens were coupled to BSA by carbodiimide reaction. Pre-incubation of anti-DEC sera with various haptens such as DEC, M P C A or piperazine citrate effectively inhibited their reactivity to m f sheath (Table 2). DEC predictably was more efficient than the other two haptens in this respect. This finding agrees with our earlier demonstration by inhibition ELISA that DEC binds with these antibodies far more efficiently than M P C A or piperazine citrate [3]. Host serum components that have been shown to get incorporated in m f [7] do not appear to be responsible for the reactivity of anti-DEC to m f sheath since preincubation of the antibodies with filariasis nonendemic human sera failed to inhibit the reaction. The above observations indicate a possible similarity or overlap of antigenic determinants between the filarial parasites and the antifilarial drug DEC. Indeed it appears to be the case, since we could demonstrate (by ELISA) antibodies reactive to M P C A - B S A in sera collected from patients of elephantiasis and tropical pulmonary eosinophilia and not in non-endemic normal sera (in preparation). Although it has been recently shown that DEC acts by activation of platelets [8], the findings reported in this communication open up newer avenues for studies on the mode of action of DEC against filarial parasites. It is premature to comment on the biological significance of the above observations. However, the anti-mf sheath activity of anti-DEC antibodies indicates that it could have far-reaching applications in the management of filariasis in human communities, since we and others have shown that antisheath antibodies have a clear protective role in the elimination of circulating microfilaria in Bancroftian [6] and Brugian [9] filariasis. Further experiments to study at the molecular level the cross-reactivity of anti-DEC to W.. bancrofti
mf and its reactivity to microfilariae of other species of filarial parasites are in progress in our laboratory.
Acknowledgements The authors are grateful to Drs. Manoj K. Das and V. R. Subramanyam for suggestions and discussions during the course of the work, Prof. L. N. Mohapatra for his encouragement and to Mr. Satyanarayana Rao for typing the manuscript.
References [1] Mackenzie, C. D. (1985) Trop. Dis. Bull 82, R I - R 3 7 . [2] Piessens, W. E and Beldekas, M. (1979) Nature 282, 845 - 847. [3] Ravindran, B., Das, D. and Patmaik, N. M. (1987) Med. Sci. Res. 15, 371- 372. [4] Gregoriadis, G. and Ryman, B. E. (1972) Biochem. J. 129, 123-133. [5] Lonngrcn, J. and Goldstein, I. J. (1978) Meth. Enzym. 50, 160 - 162. [6] Das, M. K., Subramanyam, V. R., Ravindran, B. and Pattnaik, N. M. (1987) J. Trop. Med. Hyg. (in press). [7] Maizels, R. M., Philipp, M., Dasgupta, A. and Partoni, E (1984) Parasitol. Immunol. 6, 185-190. [81 Cesbron, J. Y., Capron, A., Vargaftig, B. B., l_agarde, M., Pincemail, J., Braquet, P., Taelman, H. and Joseph, M. (1987) Nature 325, 533- 536. [91 McGreev~, P. B., Ratiwayanto, S., Tuti, S., McGreevy, M. M. and Dennis, D.T. (1980) Am. J. Trop. Med. Hyg. 29, 553-562.
Antibodies to diethylcarbamazine cross-react with microfilariae of
Wuchereria bancrofti B. Ravindran, A. K. S a t a p a t h y a n d N. M. P a t t n a i k Regional Medical Research Centre (Indian Council o f Medical Research), Bhubaneswar, lndia (Received 2 September 1987; accepted 8 Scptember 1987)
I. Summary Antibodies directed against the microfilarial sheath have been instrumental in the immune elimination of circulating microfilariae in human lymphatic filariasis. We report here that antibodies to diethyicarbamazine (DEC, the most commonly used anti-filarial drug) cross-react with the sheath of Wuchereria bancrofti microfilariae. Antibodies with reactivity to DEC were raised in rabbits by immunization with a conjugate of methylpiperazine carboxylic acid (MPCA, an acid hydrolysis product of DEC) coupled to bovine serum albumin. The reactivity of these antibodies with microfilarial sheath of W. bancrofti was demonstrated by indirect immunofluorescent assay and indirect immunoperoxidase assay. This reactivity could be effectively inhibited by pre-incubation of the antisera with different haptens such as DEC, MPCA or piperazine citrate.
strable activity against the parasites in vitro [I]. It is generally believed that DEC acts on the microfilariae in conjunction with the host's immune system [2]. We recently produced rabbit antibodies with reactivity to DEC in order to develop an immunoassay for the drug, to use as a tool for studying its mode of action [3]. During the course of further investigations it was found that antibodies to DEC (antiDEC) also reacted with microfilarial sheath of Wuchereria bancrofti. In the present paper we demonstrate the reactivity of anti-DEC towards microfilariae (mf) as shown by indirect immunofluorescent assay (IFA), indirect immunoperoxidase assay (IPA) and enzyme-linked immunoabsorbant assay (ELISA). The reactivity of anti-DEC to microfilarial sheath could be effectively inhibited by pre-incubation of anti-DEC with different haptens such as DEC, methylpiperazine carboxylic acid (MPCA) and piperazine citrate.
3. Materials and Methods 2. Introduction Diethylcarbamazine (DEC) has been in extensive use for the treatment and control of human lymphatic filariasis. Although it has been found to be very effective against the circulating microfilariae under in vivo conditions, the drug has no demonKey words." Filariasis; Wuchereria bancrofti; Diethylcarbamazine; Anti-drug antibody; Surface antigen Correspondence to: Dr. B. Ravindran, Regional Medical Research Centre (I.C.M.R.), P.O. Sainik School, Bhubaneswar 751005, India.
3.1. Animals Male albino rabbits weighing 1.5 to 2 kg were used to raise antisera. 3.2. Chemicals and reagents Diethylcarbamazine citrate and primaquine were kind gifts from Indian Drugs and Detergents, Cuttack, India and Sigma Chemicals Co., USA, respectively. Anti-rabbit immunoglobulins coupled to F1TC or peroxidase were purchased from Dakopatts, Denmark. Tetanus toxin was from Central Research Institute, Kasauli, India.
0165-2478 / 88 / $ 3.50 fc~: 1988 Elsevier Science Publishers B.V. (Biomedical Division)
7
3.3. Conjugate and antisera Detailed methods for the preparation of conjugate and raising antibodies with reactivity to DEC are described by us elsewhere [3]. Briefly, methyl piperazine carboxylic acid (MPCA - an acid hydrolysis product of DEC) was coupled to bovine serum albumin (BSA) or tetanus toxoid (TT) by carbodiimide reaction. The resultant conjugate M P C A - BSA was used to raise antibodies in rabbits and M P C A - T T was used for testing in ELISA. Purified IgG from the immunized sera after absorption with insolubilised BSA was found to react with DEC, as shown by inhibition ELISA [3]. For preparation o f MPCA-liposome, hand-shaken liposomes were prepared [4] with total hen's egg yolk phospholipids which contain about 15% phosphatidylethanolamine. The liposome suspension was mixed with M P C A and EDCI added. Under these conditions all the M P C A coupled will remain on the outermost surface of the multilamellar liposomes. One ml of MPCA-liposomes with or without complete Freund's adjuvant was used per dose for immunization of rabbits. Antisera were also raised using a similar immunization protocol against a conjugate of BSA coupled to primaquine by carbodiimide reaction [3, 5]. These sera served as controls to study the carrier molecule effect, adjuvant effect and the effect of coupling reagent namely 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide (EDCI). 3.4. W. bancrofti microfilariae Ten ml of nocturnal blood were collected in ACD from m f carriers. After removal of plasma the m f were purified by filtration through 5 ~M polycarbonate membranes (Nuclepore Inc., USA) using T r i s - E D T A buffered saline (TEBS: Tris 2 mM, EDTA 2 mM, KCI 0.3 g/l, NaCI 8.0 g/i, glucose 2.0 g/l, pH 7.2). Viable sheathed m f were washed with TEBS and used for preparation of "antigen slides" for IFA and IPA. A somatic antigen extract was also prepared through ultrasonication as described by us previously [61. 3.5. Indirect irnmunofluorescent assay (1FA) Purified m f were spotted on microslides, air-dried and fixed with acetone for 30 min and then stored at - I0 °C until further use. The microslides were examined under the microscope before performing the
assay and only slides containing 10-15 sheathed m f were used. About 15 /zl of serum or its dilutions were applied to the slides and incubated in a humid chamber for 2 h at 37 °C. The slides were then washed thrice with PBS and about 15 ~1 of 100-fold diluted swine anti-rabbit immunoglobulin-FlTC conjugate were added to the spots and incubated at 4 ° C overnight. They were then extensively washed with PBS and mounted in 30°7o glycerol in PBS and examined under a Nikon model Labophot fluorescent microscope. Since reactions to the sheath and to the cuticle are independent, care was taken to differentiate these structures under the fluorescent microscope. 3.6. Indirect immunoperoxidase assay (IPA) Essentially the procedure as described above for IFA was followed with the following differences. The antigen slides were pretreated for 20 min with 0.5°7o H 2 0 2 in methanol to inactivate endogenous peroxidase activity. Swine anti-rabbit immunoglobulin conjugated with peroxidase was used as a second antibody and the reaction was visualized under the light microscope after staining the slides with diaminobenzidine (50 mg/100 ml) in Tris-HCI buffer pH 8.8. 3.7. Inhibition of IFA reactivity with haptens Two hundred tzl of anti-DEC serum (1/4 diluted in PBS with 1°70BSA) were mixed with 200/~1 of various concentrations of DEC or M P C A er piperazine citrate in PBS, pH 7.2, and incubated at 37 °C for 1 h and then spotted on m f 'antigen slides'. The slides were then processed as mentioned above for IFA. The minimum concentration of the hapten that completely inhibits the anti-DEC reactivity to m f sheath was assessed by visual reading under the fluorescent microscope. 3.8. ELISA The detailed procedure is described elsewhere [3]. For coating, sonicated m f antigen was used at a concentration of 9/~g/ml and T T - M P C A at a concentration of 15 #g/ml in PBS. The rabbit sera were titrated and the bound antibody activity was detected by using swine anti-rabbit Ig conjugated to peroxidase (1/500 dilution). The enzyme activity was measured using O-phenylenediamine in citrate phosphate buffer, p H 5.5. After stopping the reaction with 8 N H2SO 4 the OD were measured at
492 nm using a micro-ELISA reader (Bio-Rad, USA). The highest dilution of test serum with three times or more OD as compared to normal rabbit serum was expressed as its titre.
°
°
4. Results
4.1. Reactivity with microfilariae Absorbed anti-DEC serum raised (using B S A - M P C A ) in 5 different rabbits was found to react with m f sheath o f W. bancrofti as shown by I FA and IPA (Table 1). The titre (mf reactivity) of the sera ranged from 1/8 to 1/64. The reaction was restricted only to the m f sheath and not to the cuticle (Fig. 1). Ex-sheathed parasites showed no reactivity with anti-DEC and none of the 5 pre-immunization sera reacted with m f sheath. The anti-DEC sera were tested against each of the preparations of m f collected from 10 different asymptomatic m f carriers and were found to be uniformly reactive. The rabbit (R-719) immunized with MPCA-liposomes in conjunction with CFA also showed anti-DEC activity and reacted with m f sheath (Table 1) while rabbit R-718 in which MPCA-liposomes were administered without any adjuvant failed to elicit detectable levels of anti-DEC during the period of observation. The anti m f titre of the immune sera as shown by Table 1 A n t i b o d y titres in i m m u n i z e d rabbit sera. a Rabbit No.
R-45 R-46 R-305 R-901 R-546 R-718 R-719 R-715 R-716
M a t e r i a l used b
M P C A - BSA M P C A - BSA M P C A - BSA M P C A - BSA M P C A - BSA MPCA - liposomes MPCA - liposomes P Q - BSA P Q - BSA
Batch
1 [ I I II
Anti-DEC titre ¢
Anti-mr titre c
ELtSA
IFA
IPA
6400 12800 12800 25600 25600 50 3200 <50 <50
8 64 16 8 32 NR 4 NR NR
NT 64 8 8 32 NR 4 NR NR
a Sera collected between 3 0 - 4 0 days after i m m u n i z a t i o n were used for the assays. h All the a n i m a l s except R-718 received the respective c o n j u g a t e s mixed with C F A . R e c i p r o c a l of titre. NT not tested; NR = non-reactive.
Fig. 1. Indirect i m m u n o p e r o x i d a s e assay-reactivity o f a n t i - D E C a g a i n s t microfilaria¢. (a) 100 × ; ( b ) 4 0 0 × ; (c) reactivity only to the sheath and not to the cuticle (arrow) (40 ×).
IPA or IFA was, however, very low as compared to their anti-DEC activity as revealed by ELISA (Table 1). Sera collected from two rabbits immunized with PQ-BSA showed no reactivity to m f sheath (Table 1). Affinity purified anti-DEC antibodies were found to react weakly to the surface of sheathed m f as shown by IPA and IFA. Rabbit sera (anti-DEC) collected sequentially during the course of immunization were tested by ELISA against M P C A - T T and the somatic antigen preparation of mf. While the reactivity to M P C A - T T increased very appreciably during the course of immunization, the anti-mr activity increased only 2- to 4-fold (Fig. 2). 4.2. Inhibition of anti-sheath activity The reactivity of anti-DEC to m f sheath could be effectively inhibited by pre-incubation of the sera with various haptens such as DEC, M P C A and piperazine citrate (Table 2). Pre-incubation with normal human sera, however, did not inhibit the reactivity.
Table 2 M i n i m u m c o n c e n t r a t i o n CmM) required for i n h i b i t i o n of antisheath activity ([FA). H a p t e n used
R-46
R-305
R-901
DEC MPCA P i p e r a z i n e citrate
1.25 5
1.25 5 5
1.25 5
:.L-
1.2-
E
}.0-
C'4
"7- -, -
-3'
0.6 i (D
j'
0
/ ./"
,// •. . . . . . . . . .
l~
l
Q~
..
_~×lr•.
G
~0
.:...:...
20
-. : X X T y ~ L
30
-X
--~T
~
<[
DAYS Fig. 2. I':I.ISA-anti-DEC activity of 1RS and their reactivity to somatic antigen of mf. R-546 • - . ; R - 9 0 1 • - - - * ; R - 7 1 5 x - x and R-716 x - - - x tested against M P C A - T T . R-546 • - • and R-901 • --- • tested against T T (sera diluted 100-fold for the above assays). R-546 o - o and R-901 o--- o tested against m f antigen; 1/10 diluled sera were used for testing.
5. Discussion
The present study was undertaken after our initial fortuitous observation that antibodies with reactivity to DEC (anti-DEC) could cross-react with the surface of W. bancrofti m f as shown by IPA and IFA. Detailed investigations with all our anti-DEC sera revealed their uniform reactivity to m f collected from the peripheral blood o f ten different asymptomatic carriers. The reactivity, however, was restricted only to the m f sheath and not to the cuticle. Their titre against m f sheath was very low as compared to their anti-DEC activity, notwithstanding the differences in the sensitivity of ELISA and IFA or IPA. This is further corroborated by the degree of anti10
DEC reactivity against T T - M P C A and sonicated m f antigen as revealed by ELISA (Fig. 2). Sera from rabbits immunized with a conjugate of BSA coupled to primaquine (PQ) failed to react with m f by any of the three different tests. Similarly they showed no reactivity with M P C A - B S A (Fig. 2) although their anti-PQ titre was very high in ELISA (data not shown). These animals had received quantities of BSA and CFA comparable to those of rabbits in which anti-DEC was raised using B S A - M P C A . In both cases the respective haptens were coupled to BSA by carbodiimide reaction. Pre-incubation of anti-DEC sera with various haptens such as DEC, M P C A or piperazine citrate effectively inhibited their reactivity to m f sheath (Table 2). DEC predictably was more efficient than the other two haptens in this respect. This finding agrees with our earlier demonstration by inhibition ELISA that DEC binds with these antibodies far more efficiently than M P C A or piperazine citrate [3]. Host serum components that have been shown to get incorporated in m f [7] do not appear to be responsible for the reactivity of anti-DEC to m f sheath since preincubation of the antibodies with filariasis nonendemic human sera failed to inhibit the reaction. The above observations indicate a possible similarity or overlap of antigenic determinants between the filarial parasites and the antifilarial drug DEC. Indeed it appears to be the case, since we could demonstrate (by ELISA) antibodies reactive to M P C A - B S A in sera collected from patients of elephantiasis and tropical pulmonary eosinophilia and not in non-endemic normal sera (in preparation). Although it has been recently shown that DEC acts by activation of platelets [8], the findings reported in this communication open up newer avenues for studies on the mode of action of DEC against filarial parasites. It is premature to comment on the biological significance of the above observations. However, the anti-mf sheath activity of anti-DEC antibodies indicates that it could have far-reaching applications in the management of filariasis in human communities, since we and others have shown that antisheath antibodies have a clear protective role in the elimination of circulating microfilaria in Bancroftian [6] and Brugian [9] filariasis. Further experiments to study at the molecular level the cross-reactivity of anti-DEC to W.. bancrofti
mf and its reactivity to microfilariae of other species of filarial parasites are in progress in our laboratory.
Acknowledgements The authors are grateful to Drs. Manoj K. Das and V. R. Subramanyam for suggestions and discussions during the course of the work, Prof. L. N. Mohapatra for his encouragement and to Mr. Satyanarayana Rao for typing the manuscript.
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