histolytica Proteins and the Detection of

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1 GASTROENTEROLOGY 1982;83: A Solid-Phase Sandwich Radioimmunoassay for Entamoeba histolytica Proteins and the Detection of Circulating Antigens in Amoebiasis SHIV PILLAI and ALOKE MOHIMEN Kothari Centre of Gastroenterology, The Calcutta Medical Research Institute, Calcutta, India A sensitive and specific immunochemical assay for Entamoeba histolytica antigens would be a valuable tool for clinical diagnosis, to study the sequelae of amoebiasis, and to screen for the expression of amoebic proteins in recombinant bacterial clones. The major impediment toward developing such an assay is the cross-reactivity of anti-e. histolytica antisera with a wide range of mammalian serum proteins. A rabbit anti-e. histolytica antiserum was repeatedly passed over three bovine serum protein immunoadsorbents and affinity purified over an E. histolytica protein-sepha:r;ose 4B matrix. The purified antibody was radiolabeled and formed the upper layer of two specific and sensitive sandwich radioimmunoassays for amoebic proteins. One assay used a rabbit antiamoebic antibody solid phase and the other a human antibody solid phase. The latter assay proved capable of detecting amoebic antigens in the polyethylene glycol precipitates of sera from 21 of 21 patients with amoebiasis (and none of 22 control subjects). The diagnosis of amoebiasis is often difficult to establish (1) particularly in the life-threatening complications of this disease. Although serologic tests Received February 17, Accepted July 9, Address requests for reprints to: Dr. Shiv Pillai, Kothari Centre of Gastroenterology, The Calcutta Medical Research Institute, 7/2 Diamond Harbour Road, Calcutta , India. The authors thank Dr. K. N. Jalan for his encouragment, Dr. Louis Diamond of the National Institutes of Health, Bethesda, Md. for valuable discussions and advice on axenic cultivation of E. histolytica, Dr. S. R. Das of the Central Drug Research Institute, Lucknow for the NIH:200 strain. The authors also thank Mr. Soumen Mehra for his assistance in axenic cultivation and Mr. Arjun Das and Mr. Prabir Ganguly for repeatedly providing them with their sera as a source of anti-e. histolytica antibodies. Mr. E. G. P. Nair and Mr. Arjun pas are also acknowledged for secretarial assistance and artwork, respectively by the American Gastroenterological Association / $02.50 (2) may aid in presumptive diagnosis, they cannot distinguish past from present infection and may even be negative in recently infected individuals. Occasionally, asymptomatic individuals may present with Entamoeba histolytica cysts in the stool; the need exists for a marker of disease activity. A sensitive and specific immunoassay for E. histolytica antigens would be of particular value in the following situations: (a) in establishing a clinical diagnosis of amoebiasis, (b) in studying the sequelae of E. histolytica infections in humans, (c) as a marker of disease activity, and (d) as a tool for molecular biologists studying this parasite, particularly to screen for the expression of E. histolytica proteins in recombinant bacterial clones. Entamoeba histolytica trophozoites cannot be cultivated axenically in the absence of serum (3,4) and serum proteins have been found to adhere to the plasma membranes of ax en i cally cultivated, washed trophozoites (5). Antisera against E. histolytica trophozoites contain, even after affinity purification over soluble "amoebic" protein immunoadsorbents, high titers of antibodies which cross-react with a variety of mammalian serum proteins, including human immunoglobulin G (IgG). This cross-reactivity is a major impediment toward the development of specific immunoassays for amoebic antigens. We describe here two sensitive and specific sandwich radioimmunoassays for amoebic antigens, one of which is capable of detecting circulating E. histolytica proteins in human amoebiasis. Methods Entamoeba histolytica Antigens The NIH:200 strain of E. histolytica was cultivated axenic ally in Diamond's TYI-S-33 (4) medium that contains 10% (vol/vol) bovine serum. Cells were washed four times with 50 mm potassium phosphate, ph 7.4, contain-

2 December 1982 RADIOIMMUNOASSAY FOR AMOEBIC ANTIGENS 1211 ing 150 mm NaCI (PBS), suspended in the above buffer containing 2 mm phenylmethyl sulfonyl fluoride, and disrupted using a hand-held Potter-Elvejhem type homogenizer. This homogenate was adjusted without centrifugation to an optical density of 2.0 at 280 nm with PBS and was llsed for immunization as described below. A soluble antigen fraction was prepared by centrifuging the homogenate at 4 C for 30 min at 27,000 g. The supernatant was concentrated using commercially obtained high-molecular-weight polyethylene glycol (Aquacide III, Calbiochem Behring Corp., San Diego, Calif.), and dialyzed extensively against PBS. Protein content was estimated by the method of Lowry et al. (6) using crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as standard. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 100 f.lg amounts of (a) the soluble amoebic protein mixture, (b) bovine serum albumin, and (c) bovine y-globulin (Sigma Chemical Co.) was performed by a modification of the method of Laemmli (7) as described elsewhere (8). Affinity Matrices Twenty-milligram amounts of the following proteins were coupled with 5-ml aliquots of Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) using divinyl sulfone (9) (Aldrich): (a) bovine serum dialyzed against 200 mm NaHC03, ph 9.0, (b) bovine serum albumin, (c) bovine y-globulin, (d) the soluble E. histolytica protein mixture described above, and (e) rabbit IgG purified on diethylaminoethyl (DEAE) cellulose (10). All gels were equilibrated in PBS. Paper-Disk Conjugates Whatman No. 1 filter paper (Whatman Inc., Paper Div., Clifton, N.J.) was punched into disks of 5-mm diameter. Two hundred fifty disks were suspended in 15 ml of 1 M Na2C03' ph 11.0, and while shaking gently 1 ml of divinyl sulfone was added drop by drop. After 70 min at room temperature, the disks were washed in succession with 500 ml amounts of (a) PBS, (b) distilled water, and (c) 200 mm NaHC03, ph 9.0. To different batches of activated paper disks (250Ibatch), 15 ml of l-mg/ml solutions of the following proteins in 200 mm NaHC03, ph 9.0, were added: (a) anti-e. histolytica rabbit y-globulin purified as described below, (b) antiamoebic human y-globulin, (c) bovine serum dialyzed against 200 mm NaHC0 3, ph 9.0, (d) human y-globulin (Sigma Chemical Co.) with no detectable anti-e. histolytica activity, and (e) horseradish peroxidase (Sigma Chemical Co., type 1). After shaking for 6 h at room temperature, 2 g of glycine was 'added to each batch to quench unreacted sites on the activated paper disks; after a further 30 min, the disks were washed with 1 L of 200 mm Tris-HCl, ph 7.5, containing 300 mm NaCI. The disks were stored at 4 C in PBS containing 0.02% wtl vol sodium azide and 0.5% wt/vol ovalbumin (Diluent buffer, DB). Anti-E. histolytica Immunoglobulin Equal volumes of the uncentrifuged E. histolytica homogenate and complete Freund's adjuvant were emulsified and rabbits were immunized with 2 ml amounts (half s.c., half Lm.) at 3D-day intervals. Titers of sera obtained 10 days after each booster were determined by indirect hemagglutination (11). Two milliliters of pooled, inactivated (56 C, 30 min) antiserum was passed repeatedly through 5 ml of bovine serum-sepharose 4B (2 cm-diameter). The column was washed out with excess PBS and the "unbound" antiserum was evaluated by crossed immunoelectrophoresis with an intermediate gel and by indirect binding studies described below. The y-globulin fraction of this diluted antiserum was obtained by precipitation with 33% ammonium sulfate, followed by dialysis against PBS. Since residual reactivity to bovine serum proteins (as determined by binding studies) remained, this y-globulin fraction was repeatedly passed through bovine serum albumin-sepharose 4B and bovine y-globulin-sepharose 4B columns (2-cm diameter, 5-ml bed volume). Fifteen milligrams of the unbound y-globulin was coupled to paper disks and the remainder was passed through a soluble E. histolytica protein-sepharose 4B column (1.5- cm diameter, 2-ml bed volume). The column was washed free of detectable protein with PBS and eluted with 1M NH 4 0H. The protein-containing fractions were immediately pooled and dialyzed extensively against PBS. This affinity-purified rabbit anti-e. histolytica antibody was radioiodinated (12) for use in the radioimmunoassays described below. Crossed Immunoelectrophoresis With an Intermediate Gel Crossed immunoelectrophoresis of 40 f.lg of soluble E. histolytica protein was performed according to Axelsen (13) into an intermediate agarose gel containing the antiserum purified by passage over a single immunoadsorbent, as described above, and then into a gel containing the original rabbit anti-e. histolytica antiserum (final dilution of antiserum in both gels 1:25). Indirect Binding Studies Using 125I-Labeled Anti- Goat Immunoglobulin G The anti-e. histolytica rabbit antiserum passed through a single immunoadsorbent (and corrected for dilution) and the original anti-eo histolytica rabbit antibody were both diluted in DB to a final concentration of 1:100 and O.l-ml amounts of each were added in duplicate to each of the following paper disks: (a) horseradish peroxidase disks used as a negative control, (b) human y globulin disks, and (c) bovine serum disks. After 3 h at 37 C, the disks were washed four times each with 3 ml of PBS containing 0.05% vol/vol Tween 20 (PBST) (Sigma Chemical Co.). One-tenth milliliter amounts of 125I-Iabeled affinity purified goat antirabbit IgG (5.5 f.lci/ml. sp act 0.57 mci/mg) were added to each of the disks and, after 3 h at 37 C, the disks were washed as described above and

3 1212 PILLAI AND MOHIMEN GASTROENTEROLOGY Vol. 83, No.6 counted in a well-type gamma counter with an efficiency of 40%. Direct Binding Studies One-tenth milliliter amounts of the radiolabeled, affipity-purified rabbit anti-e. histolytica antibody (8.17 Ci/ml, sp act 1.01 mci/mg) were added in duplicate to paper disks to which the following proteins had been covalently attached: (a) bovine serum proteins, (b) human y-globulin, and (c) horseradish peroxidase. After 3 h at 37 C, the disks were washed as described above and counted. Antiamoebic y-globulin Sera of patients with active amoebiasis and of laboratory personnel with a history of recurrent amoebiasis were screened for amoebic antibodies by an indirect hemagglutination assay. High-titered sera were pooled and the y-globulin fraction was obtained by 33% ammonium sulfate precipitation followed by extensive dialysis against PBS. A portion of the anti amoebic human y-globulin was affinity purified on an amoebic protein-sepharose 4B column (as described above for the rabbit antibody) and this purified antibody was radioiodinated. Solid-Phase Radioimmunoassays Using Radiolabeled Antiamoebic Antibody Two radioimmunoassays were set up, one using paper disks to which antiamoebic rabbit y-globulin had been attached, and the other using paper disks to which antiamoebic human y-globulin had been coupled. In both assay systems D.1-ml amounts of the following samples were added in duplicate to the paper disks. Samples used to test the upper limits of nonspecific binding in this assay. Samples routinely used in assays were bovine serum diluted 1:10 in DB and human y globulin (3 mg/ml in DB). In addition, other samples tested were polyethylene glycol (2.5% wt/vol; approximate mol wt 6000; Sigma Chemical Co.) precipitates prepared according to the method of Gangeot-Keros et a1. (14) from bovine serum, human albumin (5% wtlvol), and human y globulin (2% wt/vol). Soluble amoebic antigen. The soluble NIH:200 mixture was diluted in DB and added in amounts ranging from 20 ng to 4000 ng. Polyethylene glycol precipitates of human sera. 2.5% wtlvol polyethylene glycol precipitates of sera from patients with amoebiasis and controls were added. In the assay using human antibody disks, precipitates from 21 patients with active amoebiasis (11 with untreated colonic disease, 10 with liver abscess of which 2 were untreated and 8 partially treated) and 22 control subjects (17 in apparent good health, 3 with idiopathic inflammatory bqwel disease with no evidence of amoebic infection, 2 with hepatic malignancy) were assayed. Six of the patients with colonic amoebiasis were reassayed 6 wk or longer after completing a course of therapy. In the assay using rabbit antibody disks, eight of the above precipitates from amoebiasis patients (colonic 5, hepatic 3) and six from apparently healthy control subjects were assayed. Sera from patients and control subjects. Sera from the above patients and controls were assayed both neat as well as diluted 1:5 in DB. After 3 h at 37 C, the disks were washed four times with 3-ml amounts of PBST. One-tenth milliliter of the affinitypurified antiamoebic rabbit antibody (8.17 Ci/ml) was added to each tube and after a further 3 h at 37 C the tubes were washed as described above and counted. Solid-Phase Radioimmunoassays Using Radiolabeled Antiamoebic Antibody Two assays were set up as described above, one using antiamoebic rabbit y-globulin paper disks and the other antiamoebic human y-globulin paper disks. The radiolabeled antibody used however was an affinity-purified antiamoebic human antibody (3.18 Cilml; sp act 0.53 mci/mg). Results Sodium dodecyl sulfate polyacrylamide gel electrophoresis (not depicted here) demonstrated that two of the most prominent bands in the soluble amoebic protein mixture correspond to bovine serum albumin and bovine IgG. Crossed immunoelectrophoresis with an intermediate gel (Figure 1) demonstrated that repeated passage over a single bovine serum protein immunoadsorbent removed all detectable antibovine serum antibodies. However, indirect binding studies using radiolabeled goat antirabbit IgG (Table 1A) demonstrated residual reactivity toward bovine and human proteins. After further purification, direct binding studies (Table 1B) demonstrated that depletion was now virtually complete. A solid-phase sandwich radioimmunoassay utilizing antiamoebic human y-globulin paper disks and radioiodinated affinity-purified anti-e. histolytica rabbit antibodies (Figure 2) could detect as little as 40 ng of a soluble amoebic protein mixture. This assay could sharply distinguish between the polyethylene glycol precipitates of sera from patients with active amoebiasis and controls. The polyethylene glycol precipitates derived from control sera, bovine serum, human y-globulin, and human albumin as well as the untreated sera (both neat and diluted) from amoebiasis patients and from controls all gave similar "baseline" nonspecific readings. Polyethylene glycol precipitates from treated patients (the sera being obtained 6 wk or longer after completing a course of treatment) all gave similar baseline readings. To disdriminate between sera of amoebiasis

4 December 1982 RADIOIMMUNOASSA Y FOR AMOEBIC ANTIGENS 1213 Table 1. Radiolabeled Binding Studies to Assess Purification of Anti-Entamoeba histolytica Antibodies Disk Peroxidase 3.8 x 10 3 ')'"globulin 30.6 x 10 3 Bovine serum 92.6 x 10 3 B: Direct binding studies A: Indirect binding studies G Affinity-purified antibody after Original Single adsorbent passage through three antiserum depleted antiserum immunoadsorbents (cpm) (cpm) (cpm) (% binding)b 3.4 X X X X X X 10 3 G Antisera were diluted 1/100; affinity-purified 125I-labeled goat antirabbit IgG was added as the final reactant. b (cpm boundlcpm total) x c. <" patients and controls by this assay it is essential to use polyethylene glycol precipitates, suggesting that circulating antigens are present as immune complexes, The possible reasons as to why sera cannot be used directly are considered in the Discussion. Figure 3 depicts the results of a similar radioimmunoassay that utilized anti-e. histolytica rabbit y-globulin disks as the solid phase and the radioiodinated rabbit "developing" antibody as in the above method. This assay could detect 20 ng of the NIH:200 "standard" mixture but was incapable of discriminating between polyethylene glycol precipitates of sera from patients with active amoebiasis and controls. This discrepancy between the two assay systems used led to our attempting assays using the other possible combinations of these two sources of antiamoebic antibody. Both methods using the radiolabeled affinity-purified antiamoebic human antibody as described under Methods proved capable of discriminating between polyethylene glycol precipitates of patients with amoebiasis and control subjects (results not depicted). However, these assays were of low sensitivity (minimum detectable amount of NIH:200 mixture, 300 ng; no amoebiasis serumderived polyethylene glycol precipitate yielded counts three times as great or greater than the baseline control counts). The affinity of the radiolabeled human antibody used is probably considerably less than that of the corresponding rabbit antibody. Although the sensitivity of these two assays could perhaps be improved slightly by using a higher Figure 1. Crossed immunoelectrophoresis of soluble amoebic antigens (40 ILg) against a rabbit anti-e. histolytica antiserum (B) using an intermediate gel (A) containing the same antiserum after passage through a single bovine serum protein immunoadsorbent. Arrow denotes direction of first dimension run (150 V/cm, 10 C). In the second dimension (100 V/cm, 10 C) immunoprecipitates of bovine serum proteins formed in gel B alone. Table 2. Capacity of Assays to Discriminate Between Patients With Amoebiasis and Control Subjects Antiamoebic antibody Solid phase Developing Distinguishes amoebiasis from controls Yes No Yes Yes Sensitivity Good Good Poor Poor

5 1214 PILLAI AND MOHIMEN GASTROENTEROLOGY Vol. 83, No.6 /lo II.. a 0.. SI ::; '0 -.. N '2 0 x W w - if /lo :::IN i AAO z i 2 o 6 It: II: W., It o!! 0.._ 60 1II 1II N!! 0 z- ::> 0 u.. u.. 0 "" oo o.. A AMOEBIASIS TREATED CONTROLS NANOGRAMS AMOEBIC PROTEIN Figure 2. Solid-phase radioimmunoassay for amoebic antigens using an anti-e. histolytica human -y-globulin solid phase and a radio iodinated affinity-purified antiamoebic rabbit antibody as the developing reagent. (See Methods for details.) Polyethylene glycol precipitates of sera from patients with amoebic liver abscess disease (.6, n = 10), colonic amoebiasis (0, n = 11), and controls (0, n = 22) were tested. Six colonic amoebiasis patients were reassayed after treatment. Bovine serum (A) and human,,(-globulin (_) were routinely used to test the upper level of nonspecific binding, and a soluble amoebic protein mixture (e) was used as an arbitrary standard. concentration of developing antibody, it appears that assays using the more "precious" human antibody as the developing reagent are unlikely to prove of practical importance. The capacity of various assays (utilizing different combinations of solid phase and developing antibody) to discriminate between patients with amoebiasis and control subjects is summarized in Table 2. Discussion The diagnosis of amoebiasis presents major problems for a variety of reasons that were reviewed by Krogstad et ai. (1) and satisfactory techniques for diagnostic culture do not exist. The ability to demonstrate specific antigens in active amoebiasis should aid in clinical diagnosis as well as in tracing the... <II '".. ::: '" :;;,.. N,., '"'0 'Q.. N ljj ljj N :> N :::I Z Z i!!! Q. 0:: ljj ljj 0.. 1II 1II z Z 6 0 u... D ocf1:n 0.. :::I u "",.. AMOEBIASIS CONTROLS NANOGRAMS AMOEBIC PROTEIN Figure 3. Solid-phase radioimmunoassay for amoebic antigens using an anti-eo histolytica rabbit y-globulin solid phase and a radio iodinated affinity-purified antiamoebic rabbit antibody as the developing reagent. Symbols as in legend to Figure 2.

6 December 1982 RADIOIMMUNOASSAY FOR AMOEBIC ANTIGENS 1215 poorly defined sequelae of this disease. The assays described above using an affinity-purified, radioiodinated antiamoebic rabbit antibody are likely to prove applicable to body fluids and clinical material other than sera. They may also be of value in studying the molecular biology and immunochemistry of E. histolytica, particularly as a screening tool for recombinant deoxyribonucleic acid (DNA) approaches directed toward generating E. histolytica antigens in non protozoal hosts for eventual use in serodiagnosis and vaccine development. Of the four possible combinations of human and rabbit antiamoebic antibody tested (Table 2), only a sandwich between rabbit antibodies failed to detect E. histolytica antigens in immune complexes. This information strongly suggests that the complexed circulating antigens are of very restricted heterogeneity (probably a single protein), and that there is a particular sterically available antigenic determinant on this protein that is antigenic in humans but not readily so in rabbits. The relative ease with which antigens are detected by the sandwich method reported here suggests that the antigen of interest (if it is a single protein) may be a polymer containing multiple repeated determinants. Although, fortuitously, amoebic antigens in immune complexes have been available for immunochemical detection in the patients studied so far, this might not prove universally true. Preliminary attempts to dissociate these complexes and to radiolabel the proteins therein have been successful (Pillai et al., unpublished observations). It remains to be established whether or not a single amoebic protein is common to all immune complexes in amoebiasis, and whether or not dissociation approaches are likely to yield assays that are more sensitive or of greater clinical value than the sandwich assay reported here. Our attempts to purify and characterize the amoebic protein or proteins in immune complexes, if successful, should help resolve some of the speculations embodied in the above discussion. Polyethylene glycol precipitates each derived from 0.2 ml of serum were resuspended in the same volume of buffer. The immune complexes thus isolated could not be in a higher concentration than in the original sera; the inability to detect antigen in untreated sera by this sandwich assay system was therefore intriguing. Either or both of the following explanations could account for this phenomenon: (a) the 2.5% wt/vol polyethylene glycol precipitates contain mainly immune complexes and negligible amounts of free anti-e. histolytica antibodies; untreated sera from amoebiasis patients, in addition to the immune complexes, probably contain sufficient quantities of free antibodies which can inhibit the binding of these complexes to the solid-phase antiamoebic antibody; (b) precipitation with polyethylene glycol is unlikely to actually dissociate immune complexes; however this reagent, by its capacity to remove bound water from the surfaces of protein molecules, may sufficiently perturb the three-dimensional organization of the E. histolytica derived immune complexes so as to permit previously "hidden" antigenic determinants to be "seen" by the appropriate antibodies. Although circulating immune complexes are an established feature of most infectious diseases, the detection of circulating specific antigen (in these complexes) has generally proved a formidable task. Amoebiasis may now be considered with hepatitis B infection (15) and schistosomiasis (16), diseases in which the immunochemical detection of specific antigen has proved a sensitive tool for diagnosis, assessment of activity, and studying disease sequelae. Attempts are presently under way to use an invertase-antibody conjugate (17) in a parallel enzyme linked immunoassay system for E. histolytica antigens. Such an approach would be less expensive and more suitable for use in the geographic areas where amoebiasis is a major health problem. References 1. Krogstad DJ, Spencer JC, Healy GR. Current concepts in parasitology: amoebiasis. N Engl J Med 1978;248: Patterson M, Healy GT, Shabot JM. Serologic testing for amoebiasis. Gastroenterology 1980;78: Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica: Schaudinn 1903 and E. histolytica like amoebae. J ParasitoI1968;54: Diamond LS, Harlow DC, Cunnick CC. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 1978;72: Noya Gonzalez 0, Warren LG, Godh R. Serum proteins in the plasma membrane of Entamoeba histolytica. Archiv de Invest Medica (Mexico) 1980;11 (Suppll): Lowry OH, Rosebrough NJ, Farr A, et a1. Protein measurement with the Folin phenol reagent. J BioI Chern 1951;193: Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 Nature (Lond) 1970;227: Pillai S. Chemical cross-linking of a dimeric protein on a modified lectin matrix. A general probe for the chemical topology of oligomeric glycoproteins. Biochem J 1981; 193: Sairam MR, Porath J. Isolation of antibodies to protein hormones by bioaffinity chromatography on divinyl sulfonyl Sepharose. Biochem Biophys Res Commun 1976;69: Fahey JL, Terry EW. Ion-exchange chromatography and gel filtration. In: Weir DM, ed. Handbook of experimental immunology. Oxford: Blackwell Scientific Publications, 1978: Kessel J, Lewis W, Pasquel C, et a1. Indirect hemagglutination

7 1216 PILLAI AND MOHIMEN GASTROENTEROLOGY Vol. 83, No.6 and complement fixation tests in amoebiasis. Am J Trop Med Hyg 1965;14: Greenwood FC, Hunter WM, Glover JS. The preparation of l31i-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89: Axelsen NH. Intermediate gel in crossed and in fused rocket immunoelectrophoresis. Scand J Immunol 1973;2 (Sup pi 1): Gangeot-Keros L, Segond p, Capel F, et al. Detection of immune-complexes: a simple assay based on characterization of the in vivo bound C1 q (PEG-C1 q immunodiffusion test). J Immunol Meth 1978;23: Blumberg BS, Sutnick AI, London WT, et al. Current concepts: Australia antigen and hepatitis. N Engl J Med 1970; 283: Bout D, Santoro F, Carlier Y, et al. Circulating immune complexes in schistosomiasis. Immunology 1977;33: Pillai S, Bachhawat BK. Monoconjugate enzyme linked immunoassay. FEBS Lett 1978;90:51-3.