JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 1836-1841 0095-1137/91/091836-06$02.00/0
Vol. 29, No. 9
Rapid, Low-Technology Field- and Laboratory-Applicable Enzyme-Linked Immunosorbent Assays for Immunodiagnosis of Schistosoma mansoni CLAUDIO L. ROSSI,1 VICTOR C. W. TSANG,2 AND JOY B. PILCHER2* Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, 13081 Campinas, Sao Paulo, Brazil,1 and Parasitic Diseases Branch, Division of Parasitic Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 27 March 1991/Accepted 20 June 1991
Simple and rapid polystyrene- and nitrocellulose-based enzyme-linked immunosorbent assays were developed for detecting antibodies against adult Schistosoma mansoni microsomal antigens. The polystyrene test uses the Nunc Immuno Stick System. A single dilution of the antibody source being tested, the conjugate, and the substrate (3,3',5,5'-tetramethylbenzidine) are placed in tubes. Dried, antigen-coated polystyrene sticks are then exposed to the reagents by immersion. Once the sticks are sensitized, an entire assay can be completed in 8 min. Positive reactions result in a rich blue color in the substrate tube and can be distinguished with the naked eye. In the nitrocellulose-based test, a nitrocellulose sheet with antigen drawn in a line by pen is cut to produce identical strips. The ligand-binding steps and washings are performed in the troughs of incubation trays. The exposure times required for a single dilution of the antibody source being tested, the conjugate, and the substrate (3,3'-diaminobenzidine) are 5 min, 5 min, and 7.5 min, respectively. Once sensitized strips are available, an entire assay can be run in 50 min. Both techniques can assay serum or whole blood. The characteristics of polystyrene- and nitrocellulose-based techniques allow them to be used successfully in field studies and in minimally equipped laboratories. The systematic purification of microsomal antigens from adult worms of Schistosoma mansoni (MAMA) (8) has allowed the development of rapid, highly specific, and sensitive assays such as FAST-enzyme-linked immunosorbent assay (FAST-ELISA) (4) and rapid immunoblot (2) for detecting antibodies in schistosomiasis. The FAST-ELISA uses antigen-coated polystyrene beads on sticks molded to the lid of a microtiter plate. Reagents and sera are placed in microtiter plates, and the beads are exposed to reagents by immersion. Excluding antigen sensitization, an entire assay can be completed in 20 min. The assay sensitivity and specificity are 98 and 99%, respectively. The rapid immunoblot was developed from a standard immunoblot by increasing reagent concentrations and reducing incubation times. The assay can be performed in 30 min after the strips are prepared. The standard and rapid blots have the same sensitivities, and both techniques are capable of differentiating infections caused by the three species of the genus Schistosoma. Thus, the FAST-ELISA and immunoblot (standard or rapid) are utilized as screening and confirmatory assays, respectively, for schistosomiasis. The FAST-ELISA is appropriate for assaying several samples simultaneously. However, some diagnostic and field situations require simple screening tests able to assay a single serum specimen in a short period of time. This report describes the utilization of MAMA for the development of two new rapid, sensitive, and specific screening assays for schistosomiasis. The possibility of using whole blood instead of serum for both assays was investigated by using serum and blood standards. The first and more field-applicable assay is a tube variation of the FAST-ELISA that uses the Nunc Immuno Stick System. Dried, antigen-coated polysty*
rene sticks are exposed to reagents by immersion. Once the sticks are sensitized, an entire assay can be run in 8 min. The second assay is a nitrocellulose-based ELISA in which a nitrocellulose sheet with antigen dispensed in a line is cut to produce identical strips. Once sensitized strips are available, an entire assay can be run in 50 min.
MATERIALS AND METHODS Chemicals and reagents. All chemicals were reagent grade or better. Unless otherwise specified, they were obtained from Mallinckrodt, Inc., St. Louis, Mo. Tris was purchased from Schwarz/Mann, Inc., Orangeburg, N.Y. Polyoxyethylene sorbitan monolaurate (Tween 20) was acquired from Sigma Chemical Co., St. Louis, Mo. Thimerosal and 30% hydrogen peroxide were obtained from BDH Chemicals Ltd., Poole, England. EDTA was acquired from Fisher
Scientific Co., Fair Lawn, N.J. Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, and leupeptin were obtained from Boehringer Mannheim Biochemicals, Indianapolis, Ind. The 3,3',5,5'-tetramethylbenzidine (TMB)-peroxide substrate system was obtained from Kirkegaard & Perry Laboratories, Gaithersburg, Md., and 3,3'-diaminobenzidine (DAB) was from Aldrich Chemical Co., Milwaukee, Wis. Affinity-purified goat anti-human immunoglobulin G (IgG) (heavy and light chain activity) labeled with horseradish peroxidase was prepared as previously described (6). MAMA was prepared from adult worms of S. mansoni as described by Tsang et al.
(8).
Serum bank. A battery of 117 serum samples was assayed. The samples were grouped as follows: normal human sera (NHS) and whole blood from North American, nontraveler, healthy blood donors (n = 36) and sera from patients with S. mansoni infection (n = 36), Schistosoma japonicum infection (n = 5), echinococcosis (n = 4), filariasis (n = 4),
Corresponding author. 1836
VOL. 29, 1991
trichinosis (n = 4), paragonimiasis (n = 4), amebiasis (n = 4), syphilis (n = 4), cysticercosis (n = 4), Chagas' disease (n = 4), toxoplasmosis (n = 4), and hepatitis (n = 4). The toxoplasmosis and syphilis serum samples were obtained from residents of the United States who had never traveled to S. mansoni-endemic areas. The Chagas' disease and cysticercosis serum specimens were obtained from Brazilian and Peruvian patients, respectively, with no history of possible contact with schistosomes. Details of the other sera have previously been reported (4). Serum and blood standards. NHS and whole blood samples were obtained as described above. Serum standards, with values ranging from 10 to 900 U/pLl, were prepared by diluting an anti-S. mansoni reference serum pool (SMPR pool) containing 1,100 U/pul with an NHS pool. Details of the quantitation of the SMPR pool have previously been reported (4). Whole blood standards were prepared by mixing packed human erythrocytes (RBCs) that had been washed four times with 0.15 M NaCl-0.01 M Na2HPO4/NaHPO4, pH 7.2 (PBS) with the SMPR pool and the NHS pool. Sufficient RBCs were used to achieve a hematocrit of 40, the value approximating the middle of the normal range of healthy human blood. This was accomplished by mixing 60 pL1 of serum standard with 40 plI of packed RBCs to yield 100 RI of blood standard with a hematocrit of 40. In experiments requiring hemolyzed blood, packed RBCs were first lysed with deionized water, and lOx PBS was then added to RBC lysate to produce a final [NaCl] of 0.15 M. SMPR pool and NHS pool were added to 120-,ul aliquots of lysates in order to construct the whole blood standard curve. All standards were diluted in PBS containing 0.3% Tween 20 and 5% nonfat dried milk (PBS-Tw-milk). Before aliquots of blood standards were removed for the assays, the RBCs were resuspended. The polystyrene-based test. The Immuno Stick System (Nunc, Inc., Naperville, Ill.) consists of a polystyrene stick mounted in a screw cap that can be transferred from one reaction tube to another. The stick has an adsorbing surface of about 5.2 cm2 and is intended for reagent volumes of 1 ml in 1.8-ml polypropylene tubes. Each Immuno Stick System consists of the Immuno Stick accompanied by a 1.8-ml cryovial. These cryovials were used for all incubations. Because several sticks can be processed simultaneously and the stick is easy to remove from the screw cap, we were able to construct simple devices that hold several sticks at a time. The design of such manifold devices permitted the sticks to fit in reaction tubes containing serum, conjugate, and substrate in appropriate racks and then to be transferred to different reagents after the washing steps. In all ligandbinding steps, the sticks were manually agitated in the reagent tubes to promote mixing. Antigen sensitization. MAMA was diluted in 0.05 M TrisHCl-0.3 M KCl buffer (pH 8.0) containing 2 mM EDTA, 0.25 mM PMSF, 1 mg of pepstatin per liter, 1 mg of leupeptin per liter, and 1 g of thimerosal per liter. Aliquots (1 ml) of the diluted antigen were dispensed in polypropylene tubes arranged in a rack, and the sticks were immersed in the solution by turning the screw cap. The rack was placed on an orbital shaker (Beilco Biotechnology, Vineland, N.J.), and the sticks were sensitized with antigen by being mixed (speed setting, 3.5) at room temperature for 2 h. After antigen sensitization, the sticks were put in a beaker with PBS-0.3% Tween 20 (PBS-Tw), and the beaker was manually agitated for 10 s. The PBS-Tw was then replaced with deionized water, and the beaker was manually agitated for S more s before the water was decanted. The sticks were
IMMUNODIAGNOSIS OF SCHISTOSOMIASIS
1837
removed from the beaker and dried at room temperature for 6 h. Assays to determine optimal reagent concentrations. The polystyrene test was standardized by using excess amounts of all reagents except the one being tested. The conjugate and antigen titrations were performed by using 20 ,ul of SMPR pool per ml of PBS-Tw-milk because previous serum titrations showed that concentrations of serum greater than 15 pul/ml of diluent were necessary to achieve antibody excess. On the basis of conjugate titrations, conjugate excess was obtained at dilutions lower than 1:600. Conjugate diluted at 1:300 was used in all experiments requiring conjugate excess. In all experiments requiring antigen excess, MAMA was used at a concentration of 2 ,ug/ml. These conditions were based on previous studies with MAMAcoated polystyrene surfaces (4, 9) and on preliminary conjugate titration experiments using human IgG-coated polystyrene sticks. Linearity of substrate conversion. The linearity studies were performed with serum standards containing 20, 200, and 800 U/pul. For each one of these standards, the rate of substrate conversion was assayed at 1, 2.5, 3.5, and 5 min. Standard curve. The standard curve for the reaction was determined from an assay of artificial serum standards ranging from 20 to 900 U/pdl. Assay procedure. All sera were tested in triplicate, and the assays were conducted with sticks adjusted to an appropriate stick holder. All ligand-binding steps were performed by mixing, as described above, for 2.5 min. The sticks were consecutively incubated in tubes containing 1 ml of the following solutions: serum diluted at 1:100 with PBS-Twmilk, conjugate diluted at 1:300 with PBS-Tw, and TMBperoxide substrate system. After the serum incubation, the sticks were dipped up and down for 10 s in a reservoir containing PBS-Tw. The assay can also be done when the sticks are washed with tap water-0.3% Tween. After the conjugate incubation, the sticks were washed with PBS-Tw for 5 s, as described above, and with gently running deionized water for 10 s. After the substrate incubation, the sticks were removed and 0.15 ml of substrate was transferred from each tube to a well of a microtiter plate. The absorbances of the wells were measured at 650 nm with a microtiter plate spectrophotometer (Molecular Devices Corporation, Palo Alto, Calif.). The within-run and run-to-run variations of the test were determined with a serum standard at 150 U/pul. To determine the within-run variation, aliquots of 1 ml of the serum standard diluted in PBS-Tw-milk were dispensed into 10 reaction tubes, and the assays were performed as described above. The nitroceUulose-based test. (i) Application of antigen to nitrocelulose. A nitrocellulose sheet (8.5 by 16.5 cm) with a 0.2-pum pore size (Schleicher & Schuell, Keene, N.H.) was placed on a Write-on film sheet (21.5 by 26.6 cm) (3M, St. Paul, Minn.). Write-on film is commonly used as the plastic sheet used to prepare material for overhead projections. MAMA (30 ,ug/ml) in 0.05 M Tris-HCl-0.3 M KCl buffer (pH 8.0) containing 2 mM EDTA was applied in a line along the nitrocellulose sheet with a 0.80-mm-point-size Rapidograph pen (KOH-I-NOOR Rapidograph, Bloomsbury, N.J.). Lines 16.5 cm in length were obtained by using about 30 pld of the antigen solution. The nitrocellulose sheet was left for 1 h at room temperature before strips 0.3 cm in width were cut perpendicular to the antigen line with an Accutran strip cutter (Schleicher & Schuell). The strips were stored between two Write-on film sheets in a sealed plastic bag. (ii) Assay procedure. A protocol similar to that of the rapid
1838
J. CLIN. MICROBIOL.
ROSSI ET AL.
.f Co 0 LO
to co
0.0
0.5
1.0
1.5
[MAMA]
2.0 -
2.5
3.0
0
ug/ml
1
2 3 Time - min.
4
5
FIG. 1. Polystyrene test antigen titration. Polystyrene sticks were sensitized with MAMA at concentrations ranging from 0.1 to 3 F.g/ml (x axis). The rate-limiting element is the antigen concentra-. tion. The reacting serum was a pooled infection serum (SMPR pool) used at 15 ,ul/ml in PBS-Tw-milk. The conjugate used (goat antihuman IgG) was diluted 1:300 in PBS-Tw. TMB was used as a substrate. The rates of substrate conversion were measured at 650 nm (y axis). All determinations were performed in triplicate.
FIG. 2. Linearity of substrate conversion in the polystyrene test. Serum standards (20, 200, and 800 U/,ul) were prepared by diluting pooled infected sera (SMPR pool), quantitated at 1,100 U/,ul, with a normal human serum pool (NHS pool). For each of the serum standards, the rate of TMB conversion was assayed at 1, 2.5, 3.5, and 5 min. All determinations were performed in triplicate by using sticks sensitized with MAMA at 2 jig/ml and conjugate diluted at 1:300.
immunoblot described by Brand and Tsang (2) was employed. An Accutran system (Schleicher & Schuell) consisting of incubation trays and a strip washer was used for the assays. All sera were tested in triplicate, and all incubations were performed in the troughs of incubation trays containing 0.5 ml of the reagents. The trays were gently rocked back and forth during the incubation and washing steps. The rocking can be accomplished either manually or by using a rocker (Bellco Glass, Inc.). Sensitized strips were incubated with PBS-Tw-milk for 1.5 min to block reactive sites on the nitrocellulose. After the PBS-Tw-milk was aspirated, the strips were incubated with serum diluted at 1:50 in PBS-Twmilk for 5 min. Unbound serum components were removed by five 1.5-min washes with PBS-Tw at 50°C. The strips were then incubated for 5 min with a 1:350 conjugate dilution in PBS-Tw. After five 1.5-min washes, three with PBS-Tw followed by two with PBS, bound antibodies were visualized by incubating the strips with an H202-DAB substrate system for 7.5 min. After 10 1.5-min washes with water to stop the reaction, the strips were removed from the trays and affixed to a Write-on plastic sheet with adhesive tape. The strips were dried by air or with a hair dryer set on cold. The reactions were read by holding the plastic sheet in a vertical position; strips showing clearly defined brown lines were considered positive. In a vertical position, the positive and negative strips are easily differentiated. In a horizontal position, the physical imprint left on the nitrocellulose by the pen when applying the antigen produces a shadow that can cause a negative strip to be misinterpreted as a positive strip. After the completion of the reaction, the color intensities of the bands were quantified with the Visage 110 System (Bio Image, Ann Arbor, Mich.).
serum standards containing 20, 200, and 800 U/,u (Fig. 2). Additional experiments were performed with low-activity serum standards and an NHS pool to select the best substrate time. The 2.5-min incubation was chosen because at this time a good visual contrast between positive and negative reactions was obtained. The results from the assay using artificial serum standards ranging from 20 to 900 U/,ul fit in a four-parameter logistic curve (Fig. 3). In the polystyrene test, each serum specimen was tested in triplicate, and the mean activity was determined. A standard curve was used to translate the mean activity into U/,ul (Fig. 4). The assay of the serum battery showed that, except for three S. japonicum serum specimens, a color-based distinction between the serum specimens from patients with S.
RESULTS The polystyrene-based test. On the basis of antigen titra-
tion, antigen excess was achieved at concentrations greater than 1.5 ,ug/ml of diluent (Fig. 1). Linearity studies with the polystyrene test showed that the substrate conversion rates were linear for at least 5 min for
1.8 1.6 1.4
0 0 00
1.2 1.0 0.8
co0.6 0.4to. 0.2 0.0 10
100
1000
Serum antibody Concentration (U/Iu) FIG. 3. Polystyrene test standard curve for serum standards ranging from 20 to 900 U/plA. The coefficient of determination (R2) is 0.98. Refer to the legend of Fig. 2 for the preparation of serum standards. The antibody concentrations of the serum standards are on the x axis. The rates of TMB conversion, measured at 650 nm, are on the y axis. All determinations were performed in triplicate by using sticks sensitized with MAMA at 2 jig/ml and conjugate diluted at 1:300.
VOL. 29, 1991
IMMUNODIAGNOSIS OF SCHISTOSOMIASIS
1839
1,000-
800700
600500 40
i
400-
I
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140120-
4-a.
1009
8-
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I
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-
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10864-
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14 22 2 0
36
4
5
4
4
4
4
4
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4
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4
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0
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-
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Cl
clc .H U. WL
i
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HO O
X
O
c6a
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(b
.
FIG. 4. Results from the assay of serum specimens from 117 individuals by the polystyrene test. The groups of serum specimens are identified on the x axis. The number above each group is the number of serum specimens tested. On the y axis are the test results expressed in units per microliter. Each serum specimen was tested in triplicate, and the mean activity was derived. Note that the scale is not continuous.
mansoni infection and the other serum specimens tested was
obtained. The mean coefficient of variation for five repeats of this assay to determine within-run variation was 6.6%, with a range from 4.1 to 9.2%. The run-to-run variation was determined from the assays of serum standard in triplicate for 10 consecutive days. The mean coefficient of variation determined from the mean daily absorbance was 11%. The possibility of using whole blood instead of serum was tested. No significant differences were noted in standard curve reactivities when the test was performed with serum or blood standards. Moreover, hemolysis does not interfere with the test (Fig. 5). The nitrocellulose-based test. To determine the optimal
antigen concentration for this test, strips containing concentrations of MAMA ranging from 2 to 100 ,ug/ml were tested with artificial serum standards and an NHS pool. On the basis of antigen titration, a MAMA concentration of 30 ,ug/ml was selected. In this assay, all serum specimens from patients with S. mansoni infection, three serum specimens from patients with S. japonicum infection, and one paragonimiasis serum specimen were positive. All others were negative. The same S. japonicum serum specimens were positive by both polystyrene and nitrocellulose tests. Thus, except for the falsepositive result obtained with the paragonimiasis serum, all nitrocellulose results were identical to those obtained with the polystyrene test.
J. CLIN. MICROBIOL.
ROSSI ET AL.
1840
*~1.0
14 S1.2
0.8 K1.0 C0.8
< 0 4g 0.4
Vol. 29, No. 9
Rapid, Low-Technology Field- and Laboratory-Applicable Enzyme-Linked Immunosorbent Assays for Immunodiagnosis of Schistosoma mansoni CLAUDIO L. ROSSI,1 VICTOR C. W. TSANG,2 AND JOY B. PILCHER2* Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, 13081 Campinas, Sao Paulo, Brazil,1 and Parasitic Diseases Branch, Division of Parasitic Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 27 March 1991/Accepted 20 June 1991
Simple and rapid polystyrene- and nitrocellulose-based enzyme-linked immunosorbent assays were developed for detecting antibodies against adult Schistosoma mansoni microsomal antigens. The polystyrene test uses the Nunc Immuno Stick System. A single dilution of the antibody source being tested, the conjugate, and the substrate (3,3',5,5'-tetramethylbenzidine) are placed in tubes. Dried, antigen-coated polystyrene sticks are then exposed to the reagents by immersion. Once the sticks are sensitized, an entire assay can be completed in 8 min. Positive reactions result in a rich blue color in the substrate tube and can be distinguished with the naked eye. In the nitrocellulose-based test, a nitrocellulose sheet with antigen drawn in a line by pen is cut to produce identical strips. The ligand-binding steps and washings are performed in the troughs of incubation trays. The exposure times required for a single dilution of the antibody source being tested, the conjugate, and the substrate (3,3'-diaminobenzidine) are 5 min, 5 min, and 7.5 min, respectively. Once sensitized strips are available, an entire assay can be run in 50 min. Both techniques can assay serum or whole blood. The characteristics of polystyrene- and nitrocellulose-based techniques allow them to be used successfully in field studies and in minimally equipped laboratories. The systematic purification of microsomal antigens from adult worms of Schistosoma mansoni (MAMA) (8) has allowed the development of rapid, highly specific, and sensitive assays such as FAST-enzyme-linked immunosorbent assay (FAST-ELISA) (4) and rapid immunoblot (2) for detecting antibodies in schistosomiasis. The FAST-ELISA uses antigen-coated polystyrene beads on sticks molded to the lid of a microtiter plate. Reagents and sera are placed in microtiter plates, and the beads are exposed to reagents by immersion. Excluding antigen sensitization, an entire assay can be completed in 20 min. The assay sensitivity and specificity are 98 and 99%, respectively. The rapid immunoblot was developed from a standard immunoblot by increasing reagent concentrations and reducing incubation times. The assay can be performed in 30 min after the strips are prepared. The standard and rapid blots have the same sensitivities, and both techniques are capable of differentiating infections caused by the three species of the genus Schistosoma. Thus, the FAST-ELISA and immunoblot (standard or rapid) are utilized as screening and confirmatory assays, respectively, for schistosomiasis. The FAST-ELISA is appropriate for assaying several samples simultaneously. However, some diagnostic and field situations require simple screening tests able to assay a single serum specimen in a short period of time. This report describes the utilization of MAMA for the development of two new rapid, sensitive, and specific screening assays for schistosomiasis. The possibility of using whole blood instead of serum for both assays was investigated by using serum and blood standards. The first and more field-applicable assay is a tube variation of the FAST-ELISA that uses the Nunc Immuno Stick System. Dried, antigen-coated polysty*
rene sticks are exposed to reagents by immersion. Once the sticks are sensitized, an entire assay can be run in 8 min. The second assay is a nitrocellulose-based ELISA in which a nitrocellulose sheet with antigen dispensed in a line is cut to produce identical strips. Once sensitized strips are available, an entire assay can be run in 50 min.
MATERIALS AND METHODS Chemicals and reagents. All chemicals were reagent grade or better. Unless otherwise specified, they were obtained from Mallinckrodt, Inc., St. Louis, Mo. Tris was purchased from Schwarz/Mann, Inc., Orangeburg, N.Y. Polyoxyethylene sorbitan monolaurate (Tween 20) was acquired from Sigma Chemical Co., St. Louis, Mo. Thimerosal and 30% hydrogen peroxide were obtained from BDH Chemicals Ltd., Poole, England. EDTA was acquired from Fisher
Scientific Co., Fair Lawn, N.J. Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, and leupeptin were obtained from Boehringer Mannheim Biochemicals, Indianapolis, Ind. The 3,3',5,5'-tetramethylbenzidine (TMB)-peroxide substrate system was obtained from Kirkegaard & Perry Laboratories, Gaithersburg, Md., and 3,3'-diaminobenzidine (DAB) was from Aldrich Chemical Co., Milwaukee, Wis. Affinity-purified goat anti-human immunoglobulin G (IgG) (heavy and light chain activity) labeled with horseradish peroxidase was prepared as previously described (6). MAMA was prepared from adult worms of S. mansoni as described by Tsang et al.
(8).
Serum bank. A battery of 117 serum samples was assayed. The samples were grouped as follows: normal human sera (NHS) and whole blood from North American, nontraveler, healthy blood donors (n = 36) and sera from patients with S. mansoni infection (n = 36), Schistosoma japonicum infection (n = 5), echinococcosis (n = 4), filariasis (n = 4),
Corresponding author. 1836
VOL. 29, 1991
trichinosis (n = 4), paragonimiasis (n = 4), amebiasis (n = 4), syphilis (n = 4), cysticercosis (n = 4), Chagas' disease (n = 4), toxoplasmosis (n = 4), and hepatitis (n = 4). The toxoplasmosis and syphilis serum samples were obtained from residents of the United States who had never traveled to S. mansoni-endemic areas. The Chagas' disease and cysticercosis serum specimens were obtained from Brazilian and Peruvian patients, respectively, with no history of possible contact with schistosomes. Details of the other sera have previously been reported (4). Serum and blood standards. NHS and whole blood samples were obtained as described above. Serum standards, with values ranging from 10 to 900 U/pLl, were prepared by diluting an anti-S. mansoni reference serum pool (SMPR pool) containing 1,100 U/pul with an NHS pool. Details of the quantitation of the SMPR pool have previously been reported (4). Whole blood standards were prepared by mixing packed human erythrocytes (RBCs) that had been washed four times with 0.15 M NaCl-0.01 M Na2HPO4/NaHPO4, pH 7.2 (PBS) with the SMPR pool and the NHS pool. Sufficient RBCs were used to achieve a hematocrit of 40, the value approximating the middle of the normal range of healthy human blood. This was accomplished by mixing 60 pL1 of serum standard with 40 plI of packed RBCs to yield 100 RI of blood standard with a hematocrit of 40. In experiments requiring hemolyzed blood, packed RBCs were first lysed with deionized water, and lOx PBS was then added to RBC lysate to produce a final [NaCl] of 0.15 M. SMPR pool and NHS pool were added to 120-,ul aliquots of lysates in order to construct the whole blood standard curve. All standards were diluted in PBS containing 0.3% Tween 20 and 5% nonfat dried milk (PBS-Tw-milk). Before aliquots of blood standards were removed for the assays, the RBCs were resuspended. The polystyrene-based test. The Immuno Stick System (Nunc, Inc., Naperville, Ill.) consists of a polystyrene stick mounted in a screw cap that can be transferred from one reaction tube to another. The stick has an adsorbing surface of about 5.2 cm2 and is intended for reagent volumes of 1 ml in 1.8-ml polypropylene tubes. Each Immuno Stick System consists of the Immuno Stick accompanied by a 1.8-ml cryovial. These cryovials were used for all incubations. Because several sticks can be processed simultaneously and the stick is easy to remove from the screw cap, we were able to construct simple devices that hold several sticks at a time. The design of such manifold devices permitted the sticks to fit in reaction tubes containing serum, conjugate, and substrate in appropriate racks and then to be transferred to different reagents after the washing steps. In all ligandbinding steps, the sticks were manually agitated in the reagent tubes to promote mixing. Antigen sensitization. MAMA was diluted in 0.05 M TrisHCl-0.3 M KCl buffer (pH 8.0) containing 2 mM EDTA, 0.25 mM PMSF, 1 mg of pepstatin per liter, 1 mg of leupeptin per liter, and 1 g of thimerosal per liter. Aliquots (1 ml) of the diluted antigen were dispensed in polypropylene tubes arranged in a rack, and the sticks were immersed in the solution by turning the screw cap. The rack was placed on an orbital shaker (Beilco Biotechnology, Vineland, N.J.), and the sticks were sensitized with antigen by being mixed (speed setting, 3.5) at room temperature for 2 h. After antigen sensitization, the sticks were put in a beaker with PBS-0.3% Tween 20 (PBS-Tw), and the beaker was manually agitated for 10 s. The PBS-Tw was then replaced with deionized water, and the beaker was manually agitated for S more s before the water was decanted. The sticks were
IMMUNODIAGNOSIS OF SCHISTOSOMIASIS
1837
removed from the beaker and dried at room temperature for 6 h. Assays to determine optimal reagent concentrations. The polystyrene test was standardized by using excess amounts of all reagents except the one being tested. The conjugate and antigen titrations were performed by using 20 ,ul of SMPR pool per ml of PBS-Tw-milk because previous serum titrations showed that concentrations of serum greater than 15 pul/ml of diluent were necessary to achieve antibody excess. On the basis of conjugate titrations, conjugate excess was obtained at dilutions lower than 1:600. Conjugate diluted at 1:300 was used in all experiments requiring conjugate excess. In all experiments requiring antigen excess, MAMA was used at a concentration of 2 ,ug/ml. These conditions were based on previous studies with MAMAcoated polystyrene surfaces (4, 9) and on preliminary conjugate titration experiments using human IgG-coated polystyrene sticks. Linearity of substrate conversion. The linearity studies were performed with serum standards containing 20, 200, and 800 U/pul. For each one of these standards, the rate of substrate conversion was assayed at 1, 2.5, 3.5, and 5 min. Standard curve. The standard curve for the reaction was determined from an assay of artificial serum standards ranging from 20 to 900 U/pdl. Assay procedure. All sera were tested in triplicate, and the assays were conducted with sticks adjusted to an appropriate stick holder. All ligand-binding steps were performed by mixing, as described above, for 2.5 min. The sticks were consecutively incubated in tubes containing 1 ml of the following solutions: serum diluted at 1:100 with PBS-Twmilk, conjugate diluted at 1:300 with PBS-Tw, and TMBperoxide substrate system. After the serum incubation, the sticks were dipped up and down for 10 s in a reservoir containing PBS-Tw. The assay can also be done when the sticks are washed with tap water-0.3% Tween. After the conjugate incubation, the sticks were washed with PBS-Tw for 5 s, as described above, and with gently running deionized water for 10 s. After the substrate incubation, the sticks were removed and 0.15 ml of substrate was transferred from each tube to a well of a microtiter plate. The absorbances of the wells were measured at 650 nm with a microtiter plate spectrophotometer (Molecular Devices Corporation, Palo Alto, Calif.). The within-run and run-to-run variations of the test were determined with a serum standard at 150 U/pul. To determine the within-run variation, aliquots of 1 ml of the serum standard diluted in PBS-Tw-milk were dispensed into 10 reaction tubes, and the assays were performed as described above. The nitroceUulose-based test. (i) Application of antigen to nitrocelulose. A nitrocellulose sheet (8.5 by 16.5 cm) with a 0.2-pum pore size (Schleicher & Schuell, Keene, N.H.) was placed on a Write-on film sheet (21.5 by 26.6 cm) (3M, St. Paul, Minn.). Write-on film is commonly used as the plastic sheet used to prepare material for overhead projections. MAMA (30 ,ug/ml) in 0.05 M Tris-HCl-0.3 M KCl buffer (pH 8.0) containing 2 mM EDTA was applied in a line along the nitrocellulose sheet with a 0.80-mm-point-size Rapidograph pen (KOH-I-NOOR Rapidograph, Bloomsbury, N.J.). Lines 16.5 cm in length were obtained by using about 30 pld of the antigen solution. The nitrocellulose sheet was left for 1 h at room temperature before strips 0.3 cm in width were cut perpendicular to the antigen line with an Accutran strip cutter (Schleicher & Schuell). The strips were stored between two Write-on film sheets in a sealed plastic bag. (ii) Assay procedure. A protocol similar to that of the rapid
1838
J. CLIN. MICROBIOL.
ROSSI ET AL.
.f Co 0 LO
to co
0.0
0.5
1.0
1.5
[MAMA]
2.0 -
2.5
3.0
0
ug/ml
1
2 3 Time - min.
4
5
FIG. 1. Polystyrene test antigen titration. Polystyrene sticks were sensitized with MAMA at concentrations ranging from 0.1 to 3 F.g/ml (x axis). The rate-limiting element is the antigen concentra-. tion. The reacting serum was a pooled infection serum (SMPR pool) used at 15 ,ul/ml in PBS-Tw-milk. The conjugate used (goat antihuman IgG) was diluted 1:300 in PBS-Tw. TMB was used as a substrate. The rates of substrate conversion were measured at 650 nm (y axis). All determinations were performed in triplicate.
FIG. 2. Linearity of substrate conversion in the polystyrene test. Serum standards (20, 200, and 800 U/,ul) were prepared by diluting pooled infected sera (SMPR pool), quantitated at 1,100 U/,ul, with a normal human serum pool (NHS pool). For each of the serum standards, the rate of TMB conversion was assayed at 1, 2.5, 3.5, and 5 min. All determinations were performed in triplicate by using sticks sensitized with MAMA at 2 jig/ml and conjugate diluted at 1:300.
immunoblot described by Brand and Tsang (2) was employed. An Accutran system (Schleicher & Schuell) consisting of incubation trays and a strip washer was used for the assays. All sera were tested in triplicate, and all incubations were performed in the troughs of incubation trays containing 0.5 ml of the reagents. The trays were gently rocked back and forth during the incubation and washing steps. The rocking can be accomplished either manually or by using a rocker (Bellco Glass, Inc.). Sensitized strips were incubated with PBS-Tw-milk for 1.5 min to block reactive sites on the nitrocellulose. After the PBS-Tw-milk was aspirated, the strips were incubated with serum diluted at 1:50 in PBS-Twmilk for 5 min. Unbound serum components were removed by five 1.5-min washes with PBS-Tw at 50°C. The strips were then incubated for 5 min with a 1:350 conjugate dilution in PBS-Tw. After five 1.5-min washes, three with PBS-Tw followed by two with PBS, bound antibodies were visualized by incubating the strips with an H202-DAB substrate system for 7.5 min. After 10 1.5-min washes with water to stop the reaction, the strips were removed from the trays and affixed to a Write-on plastic sheet with adhesive tape. The strips were dried by air or with a hair dryer set on cold. The reactions were read by holding the plastic sheet in a vertical position; strips showing clearly defined brown lines were considered positive. In a vertical position, the positive and negative strips are easily differentiated. In a horizontal position, the physical imprint left on the nitrocellulose by the pen when applying the antigen produces a shadow that can cause a negative strip to be misinterpreted as a positive strip. After the completion of the reaction, the color intensities of the bands were quantified with the Visage 110 System (Bio Image, Ann Arbor, Mich.).
serum standards containing 20, 200, and 800 U/,u (Fig. 2). Additional experiments were performed with low-activity serum standards and an NHS pool to select the best substrate time. The 2.5-min incubation was chosen because at this time a good visual contrast between positive and negative reactions was obtained. The results from the assay using artificial serum standards ranging from 20 to 900 U/,ul fit in a four-parameter logistic curve (Fig. 3). In the polystyrene test, each serum specimen was tested in triplicate, and the mean activity was determined. A standard curve was used to translate the mean activity into U/,ul (Fig. 4). The assay of the serum battery showed that, except for three S. japonicum serum specimens, a color-based distinction between the serum specimens from patients with S.
RESULTS The polystyrene-based test. On the basis of antigen titra-
tion, antigen excess was achieved at concentrations greater than 1.5 ,ug/ml of diluent (Fig. 1). Linearity studies with the polystyrene test showed that the substrate conversion rates were linear for at least 5 min for
1.8 1.6 1.4
0 0 00
1.2 1.0 0.8
co0.6 0.4to. 0.2 0.0 10
100
1000
Serum antibody Concentration (U/Iu) FIG. 3. Polystyrene test standard curve for serum standards ranging from 20 to 900 U/plA. The coefficient of determination (R2) is 0.98. Refer to the legend of Fig. 2 for the preparation of serum standards. The antibody concentrations of the serum standards are on the x axis. The rates of TMB conversion, measured at 650 nm, are on the y axis. All determinations were performed in triplicate by using sticks sensitized with MAMA at 2 jig/ml and conjugate diluted at 1:300.
VOL. 29, 1991
IMMUNODIAGNOSIS OF SCHISTOSOMIASIS
1839
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FIG. 4. Results from the assay of serum specimens from 117 individuals by the polystyrene test. The groups of serum specimens are identified on the x axis. The number above each group is the number of serum specimens tested. On the y axis are the test results expressed in units per microliter. Each serum specimen was tested in triplicate, and the mean activity was derived. Note that the scale is not continuous.
mansoni infection and the other serum specimens tested was
obtained. The mean coefficient of variation for five repeats of this assay to determine within-run variation was 6.6%, with a range from 4.1 to 9.2%. The run-to-run variation was determined from the assays of serum standard in triplicate for 10 consecutive days. The mean coefficient of variation determined from the mean daily absorbance was 11%. The possibility of using whole blood instead of serum was tested. No significant differences were noted in standard curve reactivities when the test was performed with serum or blood standards. Moreover, hemolysis does not interfere with the test (Fig. 5). The nitrocellulose-based test. To determine the optimal
antigen concentration for this test, strips containing concentrations of MAMA ranging from 2 to 100 ,ug/ml were tested with artificial serum standards and an NHS pool. On the basis of antigen titration, a MAMA concentration of 30 ,ug/ml was selected. In this assay, all serum specimens from patients with S. mansoni infection, three serum specimens from patients with S. japonicum infection, and one paragonimiasis serum specimen were positive. All others were negative. The same S. japonicum serum specimens were positive by both polystyrene and nitrocellulose tests. Thus, except for the falsepositive result obtained with the paragonimiasis serum, all nitrocellulose results were identical to those obtained with the polystyrene test.
J. CLIN. MICROBIOL.
ROSSI ET AL.
1840
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