Antigens of Setaria digitata: cross-reaction with surface antigens of Wuchereria bancrofti microfilariae and serum antibodies of W. bancrofti-infected subjects

Bulletin of the World Health Organization, 58 (4): 649-654 (1980) Antigens of Setaria digitata: cross-reaction with surface antigens of Wuchereria bancrofti microfilariae and serum antibodies of W. bancrofti-infected subjects S. DISSANAYAKE1 & M. M. ISMAIL2 Extracts of Setaria digitata have been fractionated by DEAE-Sephadex A50 chromatography and thefractions obtained were used in the inhibition ofindirect immuno- fluorescence of Wuchereria bancrofti microfilariae. The fractions were also tested by an enzyme-linked immunosorbent assay (ELISA) techniquefor reactivity against antibodies in the serum of patients with W. bancrofti infections. The results indicate that S. digitata contains several antigenicfractions that show cross-reactivity againstfilariasis sera; two of thesefractions cross-reacted with surface antigens ofW. bancrofti microfilariae but, when tested by the ELISA technique, these antigens did not show the highest reactivity against serum antibodies. Serological diagnosis of filariasis by testing for circulating antibodies (1) does not necessarily detect active infection. Determination of circulating antigens may prove to be more relevant, but this requires characterization of antigens that evoke an antibody response. In the case of Wuchereria bancrofti, it is practically impossible to obtain sufficient filariae to extract anti- genic reagents for characterization. The wide cross- reactivity of filarial species permits the use of heteroantigens for this purpose, although this may not be an ideal solution. Extracts of Dirofilaria immitis have been extensively analysed in this respect (2-4). However, these antigens do not appear to be specific for W. bancrofti (1, 5) and D. immitis is becoming increasingly difficult to obtain. Therefore, studies on other filarial species are necessary. This communication deals with an attempt to characterize certain antigens of Setaria digitata that react with serum antibodies of filariasis subjects. The cross-reactivity of these antigens with the surface anti- gens of W. bancrofti microfilariae was also studied. The results indicate that several Setaria digitata antigens cross-react with serum antibodies and that only a fraction of these cross-react with surface anti- gens of W. bancrofti microfilariae. 1 Lecturer, Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka. 2 Head, Department of Parasitology, Medical Research Institute, Colombo, Sri Lanka. MATERIALS AND METHODS Collection of S. digitata adult worms Adult S. digitata were removed from the peritoneal cavity of cattle at the time of slaughter at the municipal abbatoir, Colombo, Sri Lanka. The worms were transported in saline to the laboratory and after repeated washing in phosphate-buffered saline (PBS, pH7.2), stored at -20 IC until use. Preparation ofextracts ofadult S. digitata worms Adult S. digitata worms were homogenized in PBS containing 1% of Tween 20. The homogenate was dialysed against 0.005 mol/litre phosphate buffer, pH8, with several changes of buffer, at 4 'C. The soluble material was extracted by centrifugation at 15 000 g for 30 min and the supernatant was stored at -20 IC. This preparation was denoted as 'crude antigen'. DEAE Sephadex A50 fractionation of the crude antigen DEAE-Sephadex A50' fractionation of the crude antigen was performed in phosphate buffer, pH8. The proteins in the crude antigen were either eluted in one fraction using 0.005 mol/litre phosphate buffer containing 0.35 mol/litre sodium chloride or fraction- ated with increasing concentrations of sodium a From Pharmacia Fine Chemicals, Uppsala, Sweden. 3984 -649- 650 S. DISSANAYAKE & M. M. ISMAIL chloride. The protein concentration in the eluates was monitored by reading the optical density at 280 nm. The protein peaks from the column were pooled, concentrated against solid sucrose, dialysed with PBS, and stored at -20 'C. Collection of blood serum from W. bancrofti - infected subjects Blood samples were collected from patients attend- ing the Anti-Filariasis Clinic, Dehiwela, Sri Lanka. All samples were tested for filarial antibodies by the indirect immunofluorescent antibody (IFA) technique using W. bancrofti microfilariae as antigen (whole and untreated) and for microfilariae in blood by the membrane filter technique. For the preparation of the pooled positive sera (PSI, PS2, and PS3), a minimum of 10 samples drawn from patients showing classical signs of filariasis (lymphangitis, elephantiasis, etc.) and who were strongly positive for serum antibodies (titre >64) by the IFA test were pooled. The filaria-negative sera used in the preparation of the pooled negative serum were from normal subjects without any previous history of filariasis and whose night blood was found to be negative for microfilariae and for filarial antibodies when tested by the IFA technique. These negative samples were drawn from subjects who were matched for age with the filariasis- positive subjects and were also from the same endemic area. Indirect immunofluorescent antibody test W. bancrofti microfilariae, isolated from infected patients, served as antigen. Serial dilutions of the test sera were made in PBS and to each 3-ml round- bottomed tube containing 100 ,ul of the diluted serum were added about 30 W. bancrofti microfilariae in 50 jAd of PBS; the mixture was incubated at 37 'C for 3 hours and overnight at 4 'C. The microfilariae were then washed in PBS and 100 pl of an appropriately diluted fluorescein isothiocyanate (FITC)-conjugated antihuman Ig(G + A + M)b was added. After incu- bation at 37 'C for 2-3 hours, the microfilariae were washed in PBS (5 times) and the fluorescence was read on a Leitz Dialux microscope. The antibody titre of the serum was expressed as the reciprocal of the highest dilution showing a definite fluorescence. Inhibition ofthe IFA test with antigens extractedfrom adult S. digitata Prior to incubation with microfilariae, the diluted sera (prepared as described above) were incubated with the antigen preparations for 3 hours at 37 'C. To this mixture were then added the microfilariae and the IFA test was performed as usual. b From Wellcome Reagents Ltd., Beckenham, England. Detection ofantibodies in sera offilariasis patients by means of enzyme-linked immunosorbent assay (ELISA) Serum antibodies of filariasis patients reacting with S. digitata antigens were detected by ELISA. The method used was that described by Voller et al. (6) but the substrate was disodium phenyl phosphate and 4-aminophenazone was used for colour development (7). In brief, antigens of S. digitata were coated on Cooke microtitration trays (V Type; Dynatech) in 0.06 mol/litre carbonate-bicarbonate buffer, pH 9.6. Coating was effected at 37 °C for 3 hours and over- night at 4 'C. The plates were washed with PBS con- taining 0.05% of Tween 20. Test sera were diluted in PBS containing 0.05% of Tween 20, 1 o of bovine serum albumin (BSA), and 1 % of normal rabbit serum (NRS). With a starting dilution of 1:20, serial dilutions were made on the antigen-coated plate and the final volume was maintained at 150 Ml. After incubation at 37 'C for 3 hours and at 4 'C for a further 1 hour, the plates were washed with PBS- Tween 20 (5 times) and 150 MI of 1:300 diluted enzyme conjugate (alkaline phosphatase conjugated with rabbit antihuman Ig using the procedure described by Voller et al. (6)) was added to each well and incubated at 37 'C for 1 hour and at 4 'C overnight. To the washed plates were then added 150 1l of 0.1 mol/litre disodium phenyl phosphate in 0.1 mol/litre carbon- ate buffer, pH 10. The plates were slowly rocked while being incubated at 37 'C for 2 hours. The enzyme activity in the plate wells was monitored by adding to each well 25 p1 of a 6 g/litre solution of 4-amino- phenazone in a 12 g/litre solution of potassium ferri- cyanide (fresh). A distinct purple coloration indicated a positive reaction. The colour of the blank, which consisted of reagent only, was yellow. At high serum concentrations, negative sera gave an amber colour and this was not considered as positive. The plates were read either visually or spectrophotometrically at 510 nm. When reading was done visually, the anti- body titre was expressed as the reciprocal of the highest dilution giving a distinct purple coloration. The plates were read within 10 min of addition of the aminophenazone/ferricyanide reagent. RESULTS Fractionation of crude antigen on DEAE-Sephadex A50 Fig. la shows the elution pattern obtained for the proteins of the crude antigen using 0.35 mol/litre NaCl in 0.005 mol/litre phosphate buffer, pH 8. About 10%o of the material applied to the column was not adsorbed at pH 8. All the proteins that were CROSS-REACTION BETWEEN FILARIAL ANTIGENS adsorbed eluted with 0.35 mol/litre NaCl in phosphate buffer. The eluted proteins were pooled, concentrated, and denoted as antigen SD2. Fig. lb shows the fractionation of the crude antigen with increasing concentrations of NaCl in phosphate buffer. Basically, 4 protein peaks were seen, derived from antigen SD2 of Fig. la. These were pooled, concentrated, and denoted as antigens SD2-1, SD2-2, SD2-3, and SD2-4. WHO 80561 Fig. 1. (a) DEAE Sephadex A50 fractionation of crude extract. Elution with 0.35 mol/litre NaCI in 0.005 mol/litre phosphate buffer, pH 8. (b) DEAE Sephadex A50 fraction- ation of the crude extract with increasing concentrations of NaCI. (1 = antigen SD1, 2 = antigen SD2-1, 3 = antigen SD2-2, 4 = antigen SD2-3, and 5 = antigen SD2-4). Indirect immunofluorescence and inhibition of the IFA test The IFA test was performed with pooled positive sera and pooled negative sera. The pooled positive sera (PSI, PS2, and PS3) had antibody titres ranging between 256 and 512 (Table 1). The pooled negative sera did not show any fluorescence at a dilution of 1:8. The antigens used in the inhibition of the IFA test were: crude antigen, DEAE-Sephadex A50 fractions SDI, SD2, SD2-1, SD2-2, SD2-3, and SD2-4. The degree of inhibition obtained with these antigens is shown in Table 1. Significant inhibition of the IFA test was obtained with the crude antigen, antigens SD2, SD2-1, and SD2-2. Antigens SD2-3 and SD24 were not active in the inhibition test. ELISA Fig. 2a and 2b show the effect of antigen concen- tration and antiserum dilution in the ELISA, with antigen SD2 and pooled positive and negative sera. With these reagents, satisfactory discrimination between positive and negative sera was obtained with 2 Mlg of antigen and 150 Ml of 1:50 to 1:100 diluted sera. The maximum antibody titre shown by the pooled negative serum was 40. Fig. 2c shows the ELISA results with antigens SD2-1, SD2-2, SD2-3, and SD2-4. The highest activity was seen with SD2-4. Antigens SD2-1 and SD2-2 showed only marginal activity. The reactivity of SD2-4 was about 80%o of that of antigen SD2. 0-5 ( a ) / 0J.1 10 100 0.5 Antigen concentration (ug/well) (b) _ e~~~~~~~~~~~I E C Lo _ 4, 0 0.01 01 1 X Antigen concentration (ug/well) 1:100 ¶5 O 1:100 1:10 1:1 Serum dilution SD2-4 (C) -,. o SD2-3 SD2-1 - SD2-2 4 -SD2-4 WHO 80562 Fig. 2. (a) Effect of antigen (SD2) concentration on ELISA reactivity. 1501AI of 1:100 diluted sera were added to each well. o-o pooled positive serum PS1; x-x pooled negative serum. (b) Effect of serum dilution on ELISA reactivity. Wells were coated with 2 lAg of antigen SD2 and 150 gAl of diluted sera added to each well. (c) ELISA reactivity of anti- gens SD2-1, SD2-2, SD2-3, and SD2-4. 1501l of 1:100 diluted sera were added to each well. z a) 1- 0 E LO co 6 (a) - _iT'D1 SD 2 oo N~~~~~O 3 4.' z z z c _0 0 0 2 32 Fraction No. 651 S. DISSANAYAKE & M. M. ISMAIL Table 1. Inhibition of IFA test with Setaria digitata antigens Protein concentration Test serum Inhibitor of inhibitor IFA titre Comments (mg/litre) PSi none - 512 positive control 1 Pooled negative none - negative negative control PSi crude antigen 2 16 inhibition PSi antigen SD1 1 512 no inhibition PSi antigen SD2 1 16 inhibition PS2 none - 256 positive control 2 PS2 crude antigen 0.5 8 inhibition PS2 antigen SD1 0.5 256 no inhibition PS2 antigen SD2 0.5 16 inhibition Pooled negative none - negative negative control PS3 none - 256 positive control 3 PS3 antigen SD2 0.5 16 inhibition PS3 antigen SD2-1 0.5 32 inhibition PS3 antigen SD2-2 0.5 64 inhibition PS3 antigen SD2-3 0.5 128 no inhibition PS3 antigen SD2-4 0.5 256 no inhibition a PS1, PS2, and PS3 were pooled positive sera. DISCUSSION The presence in Setaria species of antigens that cross-react with antibodies in sera of filariasis subjects has been reported by many investigators (for review see 1) with little or no information on the purification and/or characterization of these antigens. The present communication deals with the DEAE-Sephadex A50 fractionation of adult S. digitata extracts and the cross-reactivity of these fractions with surface antigens of W. bancrofti microfilariae and serum anti- bodies in filariasis subjects, as measured by an ELISA technique. Indirect immunofluorescence of microfilariae (with serum from the cases of clinical filariasis) demon- strated that the sera of these subjects contained antibodies to microfilarial surface antigens. Inhi- bition of this immunofluorescence permitted the detection of heteroantigens cross-reacting with microfilarial surface antigens. The absence of fluorescence in the control sera established that the microfilarial surface was not covered with antibodies of donor origin. By the ELISA technique described in this paper, normal subjects (i.e., those without a history of filariasis, with no microfilariae in the blood, and giving a negative IFA test for filarial antibodies in the serum) were found to be negative for filarial anti- bodies or when positive had only very low titres of antibody (10). On the other hand, filarial subjects (i.e., those with clinical signs of filariasis and with serum antibodies detectable by the IFA test), and also the microfilaraemic patients generally had signifi- cantly high levels of antibody (10). With the exception of very few cases, the symptomatic group was amicrofilaraemic. The results shown in Table 1 demonstrate that anti- gens derived from S. digitata inhibit the fluorescence of microfilariae of W. bancrofti, thus showing a cross-reaction with the surface antigens of these microfilariae. This inhibitory activity was seen with the crude antigen and with antigens SD2, SD2-1, and SD2-2. Antigens SD2-3 and SD2-4 were not active in this inhibition. In contrast, the highest reactivity in the ELISA was shown by antigen SD2-4; the antigens SD2-1 and SD2-2 were only marginally active in this test. 652 CROSS-REACTION BETWEEN FILARIAL ANTIGENS 653 The absorption of these ELISA-reacting antibodies (with S. digitata SD2-4 as antigen) with W. bancrofti antigens is of great importance. Unfortunately, we were unable to perform this experiment as adult W. bancrofti antigens were not available. However, the absorption of pooled positive sera with W. bancrofti microfilariae prior to use in the ELISA did not result in a significant reduction in the ELISA titre (S. Dissanayake & M. M. Ismail, unpublished data, 1978). These findings, taken together with the positive immunofluorescence of W. bancrofti microfilariae in the IFA test, indicate that the serum antibodies of fila- riasis subjects are directed against at least two types of antigen; the Setaria antigens SD2- 1 and SD2-2, reflecting one group that cross-reacts with surface antigens of microfilariae, and antigens SD2-3 and SD2-4, reflecting the second group (not cross-reacting with surface antigens of microfilariae) against which the majority of serum antibodies were reactive. The latter observation is in agreement with Weiss (8), who observed that anti-Dipetalonema viteae antibodies in the hamster reacted only with adult worm antigens and egg membranes, but never with intact micro- filariae. These observations may also imply the possibility of stage-specific antibody responses to different developmental stages of the parasite. Some evidence suggests the possibility of masking the microfilarial surface antigens. Papain treatment or sonication of microfilariae increases their reactivity in the IFA test (9), the plausible explanation being that hidden antigens are exposed by these treatments. It would be interesting to know the relation between these in vitro exposed antigens and the adult worm antigens. Preliminary studies on the characterization of the active components in S. digitata fraction SD2-4 have shown the presence of one antigenic species that reacts with serum antibodies in W. bancrofti-infected patients. This antigenic component appears to be a polymeric molecule with an apparent relative molecu- lar mass of 75 000-80 000 (S. Dissanayake & M. M. Ismail, unpublished data, 1978). Further studies on the characterization of this molecule are in progress. ACKNOWLEDGEMENTS This investigation received support from the filariasis component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. The authors gratefully appreciate the helpful criticism and advice received from Professor A. S. Dissanaike and Dr Bridget Ogilvie during the course of this work and especially during the preparation of the manuscript. R,SUMt ANTIGENES DE SETARIA DIGITA TA: REACTION CROISEE AVEC LES ANTIGENES DE SURFACE DES MICROFILAIRES DE WUCHERERIA BANCROFTI ET LES ANTICORPS SERIQUES DE SUJETS INFECTES PAR W. BANCROFTI La diagnostic serologique de l'infection A Wuchereria bancrofti est fonde sur la determination d'anticorps circu- lants A 1'egard d'antigenes filariens homologues ou hetero- logues. Afin d'obtenir des renseignements sur ces antigenes, des extraits de Setaria digitata ont e fractionnes par chromatographie sur DEAE-Sephadex A50 et la reactivite des fractions ainsi obtenues a e eprouvee A I'egard des anticorps seriques de sujets atteints de filariose, par le titrage avec immuno-adsorbant lie A une enzyme (ELISA). La reactivite croisee de ces antigenes avec les antigenes de surface des microfilaires de W. bancrofti a egalement ete etudiee par inhibition de l'immunofluorescence indirecte. Les resultats indiquent que S. digitata contient plusieurs fractions antigeniques qui presentent une r6activite croisee a 1'egard des serums de filariens; deux de ces fractions avaient une reactivite croisee avec des antigenes de surface des microfilaires de W. bancrofti, mais il s'agissait d'autres fractions que celle qui manifestait la reactivite la plus elev6e a l'egard des anticorps seriques dans l'epreuve ELISA. REFERENCES 1. AMBROISE-THOMAS, P. 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