Erythroadsorption and enzyme-linked immunoassays (eaia and elisa) for specific circulating antibodies and antigens in schistosomiasis

Ann. Immunol. (Inst. Pasteur) 1984, 135 I), 13-23

ERYTHROADSORPTION AND

ENZYME-LINKED

IMMUNOASSAYS

(EAIA AND ELISA) FOR SPECIFIC CIRCULATING ANTIBODIES AND ANTIGENS IN

SCHISTOSOMIASIS

by M. Stek, Jr Division of Tropical Public Health, Department o/Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (USA)

SUMMARY We developed a method of immunologictitration by erythroadsorption in order to assay anti-Schistosoma mansoni soluble cercarial antigen IgM antibodies and anti-S, mansoni soluble worm and egg antigen IgG antibodies. This method was compared to the ELISA technique. Results obtained using the two techniques were similar, although the erythroadsorption technique was less sensitive for identifying IgM during the acute phase of the disease. Both assay systems revealed a correlation between the level of circulating soluble antigens contained in the eggs and the number of eggs found in the stools of this particular group of patients. KEY-WORDS: Erythroadsorption, Schistosoma mansoni ; Immunoassay, ELISA, IgG, IgM, Human.

INTRODUCTION Significant progress has been made during the last few years in the development of new serologic techniques for antibody and antigen detection in parasitic diseases. The enzyme-linked immunosorbent assay (ELISA or EIA) is one of the simplest yet relatively sensitive and specific procedures in parasitic disease immunodiagnosis [5, 7, 10, 12-17]. Recently, an erythroadsorption immunoassay [9] (abbreviated EAIA in this paper) has been Manuscrit regu le 5 aoflt 1983, accept6 le 9 avril 1984. P r e s e n t address : United States Naval Medical Research Unit No. 3, c/o American Embassy, Cairo, Egypt (Arab Republic of Egypt).

14

M. STEK, Jr

described which offers a field-applicable technique readily interpreted by the naked eye which this paper compares to the sensitivity and specificity of ELISA. The EAIA employs specific concanavalin A (ConA) ligand-conjugated antibodies which are capable of binding sheep red blood cells (SRBC) by adsorption. Briefly, serum dilutions containing the circulating antibodies or antigens to be measured are permitted to react in solid phase microtitre plates previously sensitized (coated) with corresponding antigens or antibodies, respectively. After washing to remove unbound components, an appropriate ConA ligand-conjugated antibody is added. SRBC are introduced which adsorb onto the solid phase bound ligand. The SRBC not bound to the ligand conjugates settle to the b o t t o m of the round-bottom microtitre wells in the form of a pellet. The degree of pellet formation is inversely proportional to the degree of SRBC ligand adsorption, which is directly related to the a m o u n t of circulating antibody or antigen, respectively. This study was designed to evaluate the EAIA for specific circulating antibody and antigen detection and quantitation in patients with Schislosoma m a n s o n i infections. Therefore, E L I S A determinations for specific circulating anti-S, m a n s o n i soluble cercarial antigen-IgM antibody (~-SmSCIgM-Ab), anti-S, m a n s o n i soluble adult worm antigen-IgG antibody (~-SmSW-IgG-Ab), and S. m a n s o n i soluble egg antigen (SmSE-Ag) were compared with EAIA procedures to assess the relative efficacy of the latter techniques. M A T E R I A L S AND METHODS 1) 3licrolilre plates. Polystyrene microtitre plates with flat-bottom wells (Linbro Division, Flow Laboratories, Hamden, Connecticut) were used for the ELISA procedures. Roundbottom well polystyrene microtitre plates (Linbro Division, Flow Laboratories, Hamden, Connecticut) were used for the EAIA tests. 2) Plate sensitizalion. Plates were sensitized (coated) in the same manner for the ELISA and EAIA procedures modified according to the method of Voller el al. [15-17]. In the ~-SmSC-IgM-Ab assays, plates were sensitized by the addition of 200 ~1 of S. mansoni soluble cercarial antigen (SmSC-Ag) in 0.1 M sodium carbonate (pH 9.6) coating buffer (11.5 ~g of protein per ml) to the microtitre wells and incubated at 4 ~ C for 16 h (fig. la). The SmSC-Ag was prepared with S. mansoni ~-SmSC-Ig M-Ab = anti-S, m a n s o n i soluble e e r e a r i a l a n t i g e n IgM antibody~ ~ - S m S E - I g G - A b = anti-S, m a n s o n i soluble eg g a n t i g e n IgG a n t i b o d y . ~ - S m S W - I g G - A b = anti-S, m a n s o n i soluble a d u l t w o r m a n t i g e n IgG a n t i b o d y . BSA = bovine serum albumin. Co nA = e o n c a n a v a l i n A. P]AIA = e r y t h r o - a d s o r p t i o n i m m u n o a s s a y .

ELISA a s s a y. NPP PBS SmSC-Ag antigen. SmSE-Ag SmSW-Ag antigen. SRBC

= enzyme-linked immunosorbent = p-nitrophenylphosphate. = p h o s p h a t e - b u f f e r e d saline. = S. mansoni soluble cerearial = =

S. mansoni S. mansoni

s o l u b l e egg a n t i g e n . soluble adult worm

= sheep red bl ood cell.

EAIA

VS. ELISA IN SCHISTOSOMIASIS ASSAY

15

Puerto Rican strain cercariae shed from Biomphalaria glabrala snails in dechlorihated water, immobilized at 4 ~ C, centrifuged at 103 g for 15 min, dispersed in 2 ml of phosphate-buffered saline (PBS) p H 7.4 and sonieated to disrupt the cercariae. Following centrifugation at 105 g at 4 ~ C for 60 min, the supernatant containing the SInSC-Ag was removed and measured for protein content (Bio-Rad Assay, BioRa~ Laboratories, Richmond, CA). For the ~-SmSW-IgG-Ab studies, plates were sensitized with S. mansoni soluble adult worm antigen (SmSW-Ag). The sensitization was conducted by adding 200 ~1 of antigen in coating buffer (10.0 ~g of protein per ml) to the microtitre wells and incubating the plates at 4 ~ C for 16 h (fig. la). The SmSW-Ag was prepared with S. mansoni whole worms perfused from NMRI mice infected for 7 weeks. The worms were washed 3 times in PBS. A gravityconcentrated mixture of 500 worms/ml in PBS was ground in a r Ten Broeck ~ glass tissue homogenizer in an ice bath for 15 rain. The resulting suspension was centrifuged at 105 g at 4 ~ C for 60 min to obtain the SmSW-Ag preparation in the supernatant fluid. In the SmSE-Ag assays, 200 ~1 of rabbit-derived anti-S, mansoni soluble egg IgG

Figure 1. Erythro-adsorption immuno-assay (EAIA) for anti-schistosome specific circulating antibody. Schistosome Ag

Human Ab

(a) Wells sensitized (coated) with schistosomespecific antigen

(b) Dilufionsof human sera (circulating anti-schistesome antibody)a d d e d

ConA-ligand a-human Ab

(c) Con-A-ligand conjugated antihuman antiboP.y introduced

SRBC

(d) Sheep red blood cells (SRDC)added

(Plates washed 3 times with PBS/Tween between each step)

Figure 2. Erythro-adsorption immuno-assay (EAIA) for schistosome specific circulating antigen. a -schistosome Ab

(a) Wells sensitized (coated) with anti-schistosome specific antibody

Ag

(b) Dilutionsof human sera (circulating schistosomeantigen) added

ConA-iigand a-schistesome Ab

(c) Con-A-ligand conjugated antischistosomespecific anDbody introduced

SRBC

(d) Sheep red blood cells (SRBC)added

(Plates washed 3 times with PBS/Tween between each step) A n n . lmmunol. (Inst. Pasteur), 135 D, n ~ 1, 1984.

16

M. STEK, Jr

antibody (~-SmSE-IgG-Ab) was used to coat the microtitre wells (6.5 t~g of protein per ml). The plates were incubated at 37~ C for 4 h (fig. 2a). This coating antibody was produced in NZ rabbits repeatedly immunized intramuscularly with 200 ~g of Sm-SE-Ag in 0.5 ml PBS emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, Michigan). Three immunizations were given at monthly intervals followed by bimonthly boosters. Rabbit sera were collected at monthly intervals following the first boostei immunization and pooled for antibody preparation. The gamma globulin from the antisera was fractioned following the Rivanol-ammonium sulphate procedure [11]. The anti-schistosomal antibodies were collected by passage of the purified rabbit IgG through a column containing insolubilized SmSE-Ag. Adsorption, washing, and elution were carried out according to the method of Avrameas and Ternynek [2] using polyacrylamide agarose beads [8]. The SmSE-Ag used to immunize rabbits and to prepare the immunoadsorbent for specific antibody separation was prepared by a modification of the method used by Boros and Warren [4]. Briefly, iiltact eggs prepared from the livers and intestines of 7-week infected NMRI mice [6] were dispersed in PBS (50,000 eggs/ml) and gro.und in a ((Ten Broeck glass ,, tissue homogenizer in an ice bath. The resulting suspension was centrifuged at 105 g at 4 ~ C for 60 mn. The supernatant fluid containing the SmSE-Ag was measured for protein content using the (c Bio-Rad )) assay. After the plate sensitization (coating) procedure for both the antibody and antigen assays, the microtitre plates were washed thrice for 3 min with PBS containing 0.05% Tween 20 and 0.01% sodium azide (PBS/Tween). 3) Sera. Serum samples from 15 parasitologically positive patients were obtained prior to therapy. An estimate of the faecal egg load was made by quantitation of 3 faecal samples collected at daily intervals and measured for S. rnansoni eggs by the Bell technique [3]. Patients were classed as: negative=0 eggs/g; + = 1 - 4 9 eggs/g; + + = 5 0 - 9 9 eggs/g; + + + = 1 0 0 - 1 4 9 eggs/g; and + + - 4 - + > 1 5 0 eggs/g faeces. Six of the positive patients were from non-endemic areas who had acquired schistosomiasis during various periods of sojourn in endemic regions of Africa and the Middle East. Three of these patients could be described as having acute infections. In these latter 3 patients, it was estimated by history that infection occurred less than 3 months prior to diagnosis and serum collection. Nine other parasitologically ositive patients were from the indigenous populations of 3 endemic areas of frica and the Middle East (3 patients per area). Sera from 11 parasitologically negative individuals were used as controls. Five of these samples were from nonindigenous persons; 6 were from individuals living in the 3 endemic areas (2 controls per area) corresponding to the parasitologically positive patients in this study. . Following checkerboard testing to establish optimal dilutions, sera were diluted m PBS/Tween: 1/500 for specific ~-SmSC-IgM-Ab, 1/1,000 for specific ~-SmSWIgG-Ab, and 1/500 for circulating SmSE-Ag ELISA determinations. The serum samples were titreed 1/25 to 1/3,200 for the EAIA techniques (figs. lb and 2b). For optimal binding, the antibody assay plates were incubated at 37 ~ C for 3 h, while the antigen assay plates were incubated at 4 o C for 16 h. After incubation, pIates were washed 3 times as in the previous step. 4) Conjugates. Two hundred ~1 of alkaline-phosphatase-conjugated goat anti-human IgM antisera (Miles Laboratories, Elkhart, Indiana) diluted in PBS/Tween (8.4 ~g protein per ml) with 0.5% bovine serum albumin (BSA) were added to the microtitre wells and incubated at 37~ C for 3 h in the ELISA ~-SmSC-IgM-Ab assay.

EAIA

VS. ELISA IN SCHISTOSOMIASIS ASSAY

17

In the EAIA :~-SmSC-IgM Ab procedure, goat anti-human IgM antibodies (Miles Laboratories, Elkhart, Indiana) were conjugated with a ConA ligand (Miles Laboratories, Elkhart, Indiana) employing a glutaraldehyde coupling technique [9]. Briefly, antibody preparations were dialysed against 0,1 M sodium phosphate buffer (pH 6.8) at 4 ~ C for 16 h. Four mg of ConA were added to 1 ml of antibody (2 mg/ml) in 0.1 M sodium phosphate buffer (pH 6.8) followed by methyl-sD-mannoside to a final concentration of 0.1 M to protect the ConA binding sites. Thirty ~1 of a 1% glutaraldehyde solution were then carefully mixed into the conjugate solution and incubated at 20 ~ C for 4 h. Fifty ~1 of 2 M glycine were added and the solution incubated at 20 ~ C for 2 h. The solution was then dialysed against PBS at 4 ~ C for 16 h and centrifuged at 3 • l0 s g for 30 rain. An equal volume of glycerol was added prior to storage. These preparations were stored at --20 o C prior to use. Two hundred ~1 of PBS/Tween dilutions of the goat anti-human IgM antibody-ConA ligand conjugate (12.2 ~g/ml) with 0.5% BSA were added to the mierotitre wells and incubated at 37 o C for 3 h (fig. lc). With the ~-SmSW-IgG-Ab assay by ELISA, 200 ~1 of alkaline-phosphataseconjugated goat anti-human IgG antisera (Miles Laboratories, Elkhart, Indiana) diluted in PBS/Tween (9.0 ~g protein per ml) with 0.5% BSA were added to the microtitre wells and incubated at 37 ~ C for 3 h. For the EAIA ~-SmSW-IgG-Ab assay, goat anti-human IgG antibodies (Miles Laboratories, Elkhart, Indiana) were glutaraldehyde-eonjugated with ConA ligand. Two hundred ~1 PBS/Tween dilutions of this conjugate (11.5 ~g/ml) with 0.5% BSA were added to the microtitre wells and incubated at 37 ~ C for 3 h

(fig. it).

In the ELISA-SmSE-Ag assay, rabbit ~-SmSE-IgG was conjugated with alkaline phosphatase with glutaraldehyde [17] and employed at 8.0 ~g of protein per ml diluted in PBS/Tween with 0.5% BSA. Two hundred ~l of conjugate dilutions were added to each mierotitre well and the plates incubated at 37 ~ C for 3h. The EAIA-SmSE-Ag assay also employed rabbit ~-SmSE-IgG but conjugated with ConA ligand [9]. Two hundred ~l of this PBS/Tween conjugate dilution (9.5 ~g of protein per ml) with 0.5% BSA were added to the microtitre wells and the plate incubated at 37 ~ C for 3 h (fig. lc). After incubation, all plates were washed 3 times as before. 5) Substrate. In the ELISA procedures, p-nitrophenylphosphate (NPP) (Calbioehem, La Jolla, CA) was employed as substrate. One mg/ml of N P P was freshly prepared for each assay in 10% diethanolamine buffer (pH 9.8). Two hundred ~1 of this N P P dilution were added to the microtitre wells and incubated at 20 ~ C for 15 min for antibody assays and 30 min for antigen determinations. The color reactions were arrested with the addition of 25 al of 3 M NaOH. The absorption values were read through the plastic microtitre wells at wavelength 405 nm on an eight-channel photometer (Titertek Multiskan, Flow Laboratories, McLean, VA). 6) SRBC. In the EAIA procedures, SRBC were used rather than N P P substrate. The S1RBC were washed 3 times with sterile Elsever's medium, centrifuging at 600 g for 5 rain between washes. The SRBC were stored in sterile Elsever's medium and washed 10 times with PBS prior to use. Two hundred [xl of a 0.1% SRBC suspension in PBS were added to the microtitre wells and incubated at 20 ~ C for 2 h (figs. ld and 2d). The plates were read directly with the naked eye for the degree of pellet formation. The highest titre demonstrating a homogeneous layer or weakly diffuse ring in a well was recorded.

l,~

M. S'FEK, J r

R E S U L T S AND DISCUSSION Of the parasitologically positive patients, the 3 acutely infected individuals had the lowest faecal egg counts (x • • eggs/g of faeces) yet presented the highest ~-SmSC-IgM-Ab levels by ELISA (fig. 3). However, clear delineation of all 3 acute patients was not seen with the EAIA ~-SmSC-IgM-Ab procedure (fig. 4). Although the serologic procedures for specific anti-schistosomal IgM correlated well with each other (fig. 5), neither technique correlated with egg excretion. The parasitologically positive patients could be clearly separated from non-endemic area negative controls by the E L I S A ~-SmSW-IgG-Ab assay. However, some degree of overlap was seen with matched endemic area controls (fig. 6). Three of 5 non-endemic area controls (60%) and all 6 of t h e endemic area controls gave results by E A I A which might be interpreted as positive (fig. 7). If t h e positive titre level was set at 1/200, 2 parasitologically positive patients with a 2 § faecal egg load would have been falsely interpreted as negative by EAIA (fig. 7). Neither assay indicated t h a t specific anti-schistosomal IgG levels correlated with faecal egg loads (fig. 6 and 7) but, as with the IgM assays, the ELISA and EAIA IgG assays correlated well with each other (fig. 8). A general correlation could be seen between the concentration of circulating SmSE-Ag as measured by either E L I S A or EAIA and t h e faecal egg load (figs 9 and 10, respectively). No overlap was seen for circulating antigen by ELISA for the non-endemic area negative controls (fig. 9), but 2 of 5 patients (40%) demonstrated overlap which could possibly be interpreted as positive readings by EAIA (fig. 10). A direct correlation could be seen for SmSE-Ag measurements when the 2 methods were compared (fig. 11). In general, the E A I A appeared somewhat less sensitive t h a n ELISA. Anti-SRBC activity in the test and control sera could result in higher E A I A readings. This m a y be particularly possible for endemic areas where infections with other agents are probable, thus resulting in non-specific polyclonal antibody activity. A SRBC absorption step might control this problem, but it would further complicate the EAIA procedure and decrease its utility. Both assays could be interpreted by a naked eye titre reading. In this instance, the EAIA offers a slight advantage because of its relative ease of visual interpretation. For small-scale studies, where titering of sera could be readily accomplished, the EAIA woud be applicable. However, for extensive field investigations, the E L I S A would be preferred, since rapid screening of sera could be accomplished with a single dilution read on a photometer. In summary, complete evaluation of sera should include antibody and antigen determinations, whether by EAIA, ELISA or another serologic method. This is especially true for studies in endemic areas where large

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numbers of individuals have had previous exposure to hyperendemic diseases such as schistosomiasis, and therefore are likely to possess antibody. Because of the possibility of other parasitic infections, significant portions of the population undoubtedly possess cross-reacting antibody. Generally, serodiagnostic and seroepidemiologic studies have dealt with total antibody or IgG class assays. These are sufficient for the diagnosis of non-endemic area patients, as was seen in this study. However, these assays do not suffice for endemic area applications. Serologic analyses would be improved by inclusion of specific IgM determinations which, when elevated, have been shown to be indicative of acute infections. Circulating antigen quantitation adds another dimension to serologic studies. The assay for egg antigen by E A I A and ELISA has specificity and appears to be a means of estimating schistosomiasis infection intensity as demonstrated in this group of relatively young patients. The EAIA and ELISA have seroepidemiologie as well as serodiagnostic applications. In epidemiological terms, t h e IgG antibody assays could be used to estimate prevalence, while the IgM antibody assay and circulating antigen determinations could be employed as incidence measurements.

Rt~SUMI~ D O S A G E PAR I~RYTHROADSORPTION (EAIA) ET PAR IMMUNOENZYMOLOGIE D'ANTICORPS ET D'ANTIGI~NES SPI~CIFIQUES DANS LA SCttISTOSOMIASE

(ELISA)

Nous avons mis au point une m6thode de titrage immunologique par 6rythroadsorption afin de doser les anticorps IgM anti-Schislosoma mansoni (antigOne cercaire soluble), anticorps IgG anti-S, mansoni (antigbnes solubles contenus dans les vers et dans les ceufs). Cette m6thode a 6t6 compar6e ~ la technique de dosage enzymo-immunologique. Les r6sultats obtenus par les deux techniques sont similaires, bien que la technique par 6rythroadsorption soit moins sensible pour l'identification des IgM pendant la phase aigu~ de la maladie. Les deux systbmes de dosage ont permis de d6montrer une corr61ation entre les taux circulants d'antig6nes solubles contenus dans les oeufs et le nombre d'oeufs trouv6s dans les selles de ce groupe particulier de malades. MOTS-CLI~S : l~rythroadsorption, Schislosoma mansoni; Immunodosage, ELISA, IgG, IgM, Homme. ACKNOWLEDGEMENTS

This work was supported by the Naval Medical Research and Development Command, Work Units, No. MR041.05.01.0023, M0095PN002.5051, and the Office of Naval Research Contract No. N00014.76.C.0146. The opinions or assertions

EAIA VS. ELISA IN SCHISTOSOMIASIS ASSAY

23

contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Uniformed Services University, the U. S. Navy Department, or the Department of Defense at large. The author gratefully acknowledges the critical review and suggestions offered by Drs. Wilton Vannier and Patricia Minard and the secretarial/editorial hel t) extended by Ellen Klein.

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[3] [4]

15] [6] [7]

[8] [9] [10] [11]

[12] [13]

[14] [15] [16]

[17]

of the conjugates for the detection of antigens and antibodies. Immunochem., 1969, 6, 43-53. AVRAMEAS,S. • TERNYNCK, T., The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochem., 1969, 6, 53-66. BELL, E. R., A new method for counting Schistosoma mansoni eggs in faeces: with special reference to therapeutic trials. Bull. Org. mond. Sant& 1963, 29, 525-530. BoRos, D. L. & WARREN, K. S., Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. exp. Med., 1970, 132, 488-507. BOUT, D., DIGIMONT, J. C., FARAG, H. & CAPRON, A., hnmunodiagnosis of human parasitic diseases by ELISA, in (( Immunoenzymatic techniques ), (G. Feldman) (p. 175-182). North-Holland Publ. Co., Amsterdam, 1976. DRESDEN, M. H. ~r PAYNE, D. C., A sieving method for the collection of schistosome eggs from mouse intestines. J. Parasit., 1981, 67, 450-452. ENGVALL, E. ~ PERLMANN, P., Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochem., 1971, 8, 871-874. GUESDON, J.-L. dr AVRAMEAS, S., Polyacrylamide agarose beads for the preparation of effective immunoadsorbents. J. immunol. Methods, 1976, 11, 129-133. GUESDON,J.-L. 3r AVRAMEAS,S., Sensitive titration of antibodies and antigens using erythro-immunoassay. Ann. Immunol. (Inst. Pasteur), 1980, 131 C, 389-396. HILLYER, G. V. 8r GOMEZ DE RIOS, I., The enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of schistosomiasis. Amer. J. trop. Med. Hyg., 1979, 28, 237-241. HOREJST, J. & SMETANA, R., The isolation of gamma-globulins from blood serum by Rivanol. Acta reed. scand., 1956, 155, 65-70. HULDT, G., LARGERQUIST, B., PHILLIPS, T., DRAPER, C. C. & VOLLER, A., Detection of antibodies in schistosomiasis by enzyme-linked immunosorbent assay (ELISA). Ann. lrop. Med. Parasit., 1975, 69, 483-488. McLAREN, M., DRAPER, C. C., ROBERTS, J. M. el al., Studies on the enzymelinked immunosorbent assay (ELISA) test for Schistosoma mansoni. Ann. lrop. Med. Parasil., 1978, 72, 243-253. STEK, M., Jr, Micro-ELISA for antibody and antigen detectiou in schistosomiasis. Proc. 4th Inl. Con.q. Parasil., 1978, E, 93-04. VOLLER, A., BARTLETT, A. & BIDWELL, D. E., Enzyme immunoassays for parasitic diseases. Trans. r6y. Soc. trop. Med. Hyg., 1976, 70, 98-106. VOLLER, A., BIDWELL, D. E. & BARTLETT, A., Enzyme immunoassays for parasitic medicine. Bull. Ory. mond. Sanld, 1976, 53, 55-56. VOLLER, A., BID~VELL,D. E., BARTLETT, A. & EDWARDS, R. A., Comparison of isotopic and enzyme-immunoassays for tropical parasitic diseases. Trans. roy. Soc. trop. Med. Hyg., 1977, 71, 431-437.