JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1979, p. 778-785 0095-1137/79/12-0778/08$02.00/0

Vol. 10, No. 6

Evaluation of Enzyme-Linked Immunosorbent Assay for the Serodiagnosis of Amebiasis JAMES YANG* AND MARIE T. KENNEDY Ontario Ministry of Health, Laboratory Services Branch, Serology Section, Toronto, Ontario M5W 1R5, Canada Received for publication 20 August 1979

This report describes the development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Entamoeba histolytica. Highly sensitive and reproducible results were obtained in antigencoated plates prepared by air-drying at 370C. Comparison of the ELISA with indirect fluorescent antibody and indirect hemagglutination techniques showed that the former was slightly more sensitive than the two latter methods. The specificity was evaluated by testing specially chosen population groups. ELISA was negative in 96.4% of 693 normal adults and children and in 96.6% of 377 patients with various parasitic, bacterial, mycotic, and other clinical diseases. The assay was positive in 26% of 461 patients with suspected amebiasis and in all of 53 patients with amoebic liver abscess. The ELISA was found to be a specific, highly sensitive, and reliable procedure for detecting anti-E. histolytica antibodies in humans.

The enzyme-linked immunosorbent assay (ELISA) developed independently in 1971 by Engvall and Perlman (3) and by Van Weeman and Shuurs (14) is based on the same principles as radioimmunoassay, except that an enzyme is used as a label for the antigen or antibodies instead of a radioisotope. In the past several years, the ELISA has been successfully applied to the detection of a variety of antibodies and antigens in bacteriology, virology, parasitology, and clinical chemistry (11, 16, 18). More recently, the usefulness of the ELISA for the immunodiagnosis of amebiasis has been reported (2, 4), and the sensitivity of the assay was found to be comparable to that of the radioimmunoassay (17). We have investigated the application of the ELISA in the diagnosis of amebiasis. After analyzing the parameters of the assay and developing a standard micro-ELISA system, we compared the sensitivity of the assay with that of the indirect fluorescent antibody (IFA) and indirect hemagglutination (IHA) tests. Specificity was evaluated on specially chosen population groups. This paper describes the standard microELISA technique and its reliability and application for the detection of antibodies to E. histolytica. MATERIALS AND METHODS Antigen. The strain of Entamoeba histolytica used in this study was IP-106 (5) obtained through the courtesy of E. Meerovitch from the Institute of Par-

asitology, McGill University, Montreal, Canada. The strain was axenically grown in Diamond TPS-1 medium. For preparation of soluble antigen the amoebae were washed three times with phosphate-buffered saline (pH 7.2). After the final washing, about 5 ml of distilled water was added to each ml of packed organisms, and the amoebae were disrupted for 3 to 5 min in an ice water bath with a sonicator cell disruptor W375 (Ultra Sonico Inc., Plainview, N.Y.) with an operating frequency of 20 kHz at an output of 40 W. Finally, soluble antigen was separated by centrifuging the sonic extract at 9,000 x g for 1 h at 4°C. The soluble antigen was stored in 0.5-ml quantities at -70°C. The protein content of the soluble antigen was determined by the method of Lowry et al. (7). The same strain was used as antigen in the IFA test. Sera from clinical cases of amebiasis (53). The sera of patients suffering from amoebic liver abscess were obtained through the courtesy of investigators in Taiwan, Thailand, South Africa, Mexico, and Toronto General Hospital, Toronto. Sera from cases of suspected amebiasis (461). In this group, 172 sera were obtained from a hospital laboratory in Toronto, and the other 289 were selected from those referred for amebiasis testing to a reference laboratory. The sera had been tested by the IHA technique at the respective laboratories. Sera from patients with other diseases (377). This group had positive serological tests for various infections and conditions. There were 132 sera from patients with parasitic infections (toxoplasmosis, malaria, trichinosis, echinococcosis), 103 from bacterial infections (streptococcemia, brucellosis, whooping cough, syphilis, gonorrhea, fungi), 92 from cases of rheumatoid arthritis, and 50 from cases of hypergam-

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Sera from presumed healthy persons (693). In this group, 150 sera were obtained from pregnant women examined for prenatal Rh factor, 255 from both sexes who were screened routinely for syphilis, 104 from young women (17 to 25 years) who underwent routine check-up for gonorrhea, and 86 employees of both sexes. In addition, 98 sera from children (1 to 9 years) were also examined. Antigen carriers. Four types of microtiter plates and one type of tube were tested as antigen carriers. They were: Linbro, no. IS-FB-96S, polystyrene flat plate (Flow Laboratories, Rockville, Md.); Linbro, no. IS-MRC-96, polystyrene U plate (Flow Laboratories); Cooke, no. 1-220-25A polystyrene U plate (Dynatech Laboratories, Alexandria, Va.); Cooke, no. 1-223-29 polystyrene flat plate (Dynatech); disposable polystyrene tube, 12 by 75 mm (Falcon 2038, Falcon Plastics, Oxnard, Calif.). Conjugate. Horseradish peroxidase labeled to goat anti-human immunoglobulin G, Fab fraction (Miles Laboratories, Elkhart, Ind.), was used as a conjugate. A working dilution of the conjugate was determined by the box titration method. Optimal dilution varied from batch to batch, ranging from 1:500 to 1:2,000 of the conjugate dilutions. Substrate solution. Eighty milligrams of 5-aminosalicylic acid (K & K Laboratories, Plainview, N.Y.) was dissolved in 100 ml of hot distilled water (65 to 70°C) and kept at 4°C. The 5-aminosalicylic acid solution was brought to room temperature, and immediately before use the pH was adjusted to 6.0 with 1 N NaOH. To 9.9 ml of the 5-aminosalicylic acid, 0.1 ml of 0.5% H202 was added. ELISA. Unless otherwise stated, the following procedure was used throughout this investigation. (i) Antigen coating. A 0.05-ml amount of antigen dilution was added to each well of microtiter plates and air-dried overnight at 37°C. Triple-distilled water was used as an antigen coating solution. The dried antigen plates were then stored in a plastic bag at -200C. (ii) Performance of ELISA. Before testing, the antigen plates were washed three times with 0.9% saline containing 0.05% Tween 20. A 0.05-ml volume of an appropriate dilution of test sera was added to the wells and incubated overnight (16 to 18 h) in a moist chamber at room temperature. Known positive and negative sera were always included in each plate as controls. For the positive controls we used one strong (IFA, 1:1,280; IHA, 1:4,096) and one weak (IFA, 1:40; IHA, 1:128-256) serum. All sera were diluted in phosphate-buffered saline with 0.5% bovine serum albumin and 0.05% Tween 20 (pH 7.4). After washing as above, 0.05 ml of conjugate, diluted in the phosphate-buffered saline-0.5% bovine serum albumin-0.05% Tween 20, was added to each of the wells and incubated at 37°C in a moist chamber for 1

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using a self-emptying microcuvette (Beckman/Spinco 151). ELISA results were expressed either as a net OD value (OD of test sera minus OD of negative control), when single dilutions (1:100) of the samples were tested, or as an endpoint titer, when serial twofold dilutions were used. If a titration was used, the endpoint was the highest serum dilution that showed an OD value of l0.1. EFA test. The test was carried out as previously described by Ambroise-Thomas and Kien-Truong (1). The stained slides were read under a Leitz Orthoplan UV microscope with fluorescein vertical illumination or a Ziess microscope with an epi-fluorescence system.

RESULTS Determination of optimal antigen concentration. For this study, plates were sensitized by incubating overnight at 4°C with varying concentrations of antigen in 0.05 M carbonate buffer (pH 9.6) and tested against the two positive control sera. Best results were obtained at between 7 and 40 jig of antigen per ml (Fig. 1). At higher concentrations of the antigen there was a slight decrease in the ELISA value. Therefore, to conserve the antigen, a concentration of 7 Ag of antigen per ml was used for preparation of antigen plates in subsequent experiments. Selection of antigen carrier. In this evaluation, wells of plastic plates were treated with a 0.1-ml volume of each reagent (antigen, serum, conjugate) except substrate (0.2 ml), and tubes were treated with l.0-ml volumes of all reagents. The Cooke polystyrene plates with flat and U bottoms appeared to bind antigen more uniformly. Although no marked variation in serum titers was observed, the other types of plates gave less satisfactory results due to uneven readings. Good results were obtained with the plastic

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The wells were washed again and rinsed with distilled water. To each well, 0.2 ml of substrate solution was added, and the plates were kept at room temperature. The reaction was stopped after 30 min of incubation by adding 0.025 ml of 1 N NaOH to each well. Optical density (OD) of the reaction product was determined at 449 nm with a Spectro-Colorimeter,

ANTKIEN CONCENTRATION, .t1J1

FIG. 1. Effect of antigen concentration for optimal coating of microplates. OD at 449 nm in 30 min. (0) Strong positive serum (1:20,000); (0) weak positive serum (1:100).

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tubes, but the use of the tubes required more reagents and handling time than the plates. In our further study we therefore chose Cooke polystyrene plates with flat bottoms. Determination ofmaximum antigen coating procedure. To determine maximum binding of the amoeba antigen to the plastic surface, optimal dilution of the antigen was prepared in triple-distilled water and 0.05 M carbonate buffer (pH 9.6). Microplates with each of the preparations were then either incubated overnight at 40C in a moist chamber (wet antigen plate) or air-dried overnight at 37°C in an incubator (dry antigen plate). Figure 2 illustrates the mean values obtained from six assays on the weak positive control serum with the four types of antigen-coated plates. In the wet antigen plates, the distilled water was slightly less effi-

cient as a coating solution than the carbonate buffer, whereas in the dried plates the distilled water was equally effective, with ELISA values similar to those obtained with the carbonate buffer. However, there was a significant difference in sensitivity of the assay between the wet and dry systems for sensitization of the plates. The endpoint titer of the control serum was 1:640 in the dry antigen plates, whereas it was less than 1: 160 in the wet ones. The increased sensitivity was observed in further experiments with other positive sera. By using the dry antigen plates the sensitivity could be increased two- to eightfold higher than with the wet antigen plates (Fig. 3). The negative serum, on the other hand, remained nonreactive in both types of the antigen plates. Moreover, the dry antigen plates could

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FIG. 2. Effect of coating procedures represents mean value of six tests.

on the maximum

binding of the antigen to the microplates. Each line

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be stored either at -20°C or room temperature for a year or longer without loss of sensitivity (Table 1). In a subsequent study, all plates were sensitized by using the antigen diluted in tripledistilled water and air-drying overnight at 37°C in an incubator. Effect of reagent volume in the ELISA. In this experiment ELISA was performed by using 0.05 ml and 0.1 ml of all reagents (antigen, serum, conjugate), except the enzyme substrate, which was kept constant at 0.2 ml. Although OD values obtained with the 0.05-ml volume were approximately half of those with the 0.1-ml volume, serum endpoints differed little (Fig. 4). From these results an initial serum dilution of 1:100 and 0.05-ml reagent volumes were chosen, because the difference in OD values between the weak positive and negative sera could easily be differentiated with this system. Reproducibility. Figure 5 shows the reproducibility of the ELISA, which was determined by testing the two positive controls on 24 different days over a period of 6 months. The endpoint titers of the weak positive serum ranged from 1: 200 to 1:400, and the strong positive serum titers ranged from 1:6,400 to 1:12,800 between day-today tests. When all OD readings obtained from single dilutions of the positive (1:100 for weak and 1:3,200 for strong) and one negative sera were plotted, variations of the OD values were within 95% confidence limits during the 6-month testing period (Fig. 6). ELISA on sera from normal subjects. After optimal conditions for the ELISA system aj00

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had been established, the assay was performed on 693 sera from groups of presumably normal persons. All specimens were screened at single dilutions of 1:100. Of the sera tested, 668 (96.4%) gave OD values of 50.1, 19 (2.7%) gave values of between 0.1 and 0.19, 4 (0.6%) fell between 0.2 and 0.29, and 2 (0.3%) gave OD values of between 0.3 and 0.39. When the IFA test was performed on the 25 sera that gave OD values of >0.1 and on a random sample of 50 sera which showed OD values of