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10.1128/CDLI.7.5.810-812.2000. 2000, 7(5):810. DOI: Clin. Diagn. Lab. Immunol. Jorge-Luis de-la-Rosa Alberto Gómez-Priego, Lidia Crecencio-Rosales and Detection in Human Trichinellosis Immunosorbent Assay for Antibody Enzyme-Linked - Immunoassay Serological Evaluation of Thin-Layer http://cvi.asm.org/content/7/5/810 Updated information and services can be found at: These include: REFERENCES http://cvi.asm.org/content/7/5/810#ref-list-1 This article cites 20 articles, 2 of which can be accessed free at: CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on May 14, 2014 by guest http://cvi.asm.org/ Downloaded from on May 14, 2014 by guest http://cvi.asm.org/ Downloaded from

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  10.1128/CDLI.7.5.810-812.2000.

2000, 7(5):810. DOI:Clin. Diagn. Lab. Immunol. Jorge-Luis de-la-RosaAlberto Gómez-Priego, Lidia Crecencio-Rosales and Detection in Human TrichinellosisImmunosorbent Assay for Antibody

Enzyme-Linked−Immunoassay Serological Evaluation of Thin-Layer

http://cvi.asm.org/content/7/5/810Updated information and services can be found at:

These include:

REFERENCEShttp://cvi.asm.org/content/7/5/810#ref-list-1This article cites 20 articles, 2 of which can be accessed free at:

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/00/$04.0010

Sept. 2000, p. 810–812 Vol. 7, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Serological Evaluation of Thin-LayerImmunoassay–Enzyme-Linked Immunosorbent Assay

for Antibody Detection in Human TrichinellosisALBERTO GOMEZ-PRIEGO,1,2 LIDIA CRECENCIO-ROSALES,1 AND JORGE-LUIS DE-LA-ROSA1*

Departamento de Zoonosis, Instituto Nacional de Diagnostico y Referencia Epidemiologicos, Secretarıa de Salud,1

and Departamento de Microbiologıa y Parasitologıa, Facultad de Medicina,Universidad Nacional Autonoma de Mexico,2 Mexico City, Mexico

Received 7 February 2000/Returned for modification 5 April 2000/Accepted 19 May 2000

A new immunoenzymatic test, named the thin-layer immunoassay–enzyme-linked immunosorbent assay(TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretionproducts prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive musclebiopsies or symptoms compatible with the disease (n 5 8 or 26, respectively), all reactive in enzyme-linkedimmunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people,were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes byadding dots of 10 ml each of antigen (7 mg/ml) followed by adding diluted serum and the conjugate. Finally,the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detectedby the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameterswere 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80,respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lowerquantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific,the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment neededfor ELISA and EITB and in field studies for antibody detection in human trichinellosis.

Trichinellosis is a worldwide zoonotic infection caused byTrichinella spiralis and also by other Trichinella species (10, 22).In humans, it usually appears as epidemiologically definedoutbreaks caused by the ingestion of uncooked mammalianinfected meat (11, 17, 25, 27). Clinical symptoms are nonspe-cific, since they are similar to those present in other infectiousdiseases, resulting in a number of undetected cases. Further-more, since parasitoscopical diagnosis is frequently impracticalbecause it requires a microscopic search for larvae in musclebiopsy samples, antibody detection is the best option. Serolog-ical tests use surface, somatic, or metabolic T. spiralis antigenicproducts that are either crude, purified, or semipurified (3, 7,14, 26). Almost all procedures that detect antibodies (i.e.,complement fixation, precipitation, hemagglutination, floccu-lation, intradermal reactions, and immunofluorescent, radio-metric, and immunoenzymatic tests) have been used for theserodiagnosis of this parasitic infection (7–9, 14, 18–20). For along time, the bentonite flocculation test with excretion andsecretion (E/S) antigens, indirect hemagglutination with so-matic, semipurified extracts (Melcher’s antigen), and the im-munofluorescent-antibody test were considered the most sen-sitive, specific, and useful assays (20). Nowadays, however, themost broadly accepted procedures for diagnosis are the en-zyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunoelectrotransfer blot assay (EITB) used with E/Santigens prepared from T. spiralis muscle larvae (ML). Bothtests present good sensitivity and specificity values as demon-strated by an adequate correlation with the presence of MLboth in human patients and in naturally or experimentallyinfected animals (1, 7, 9, 21, 24, 26). Nevertheless, they require

delicate, sophisticated, or expensive equipment, such as a mi-croplate reader or electrophoresis and protein transfer equip-ment. A serological test which does not require such equip-ment, named the thin-layer immunoassay (TIA), has beenpreviously reported (12). In this test, the exposed epitopes ofthe antigen adsorbed on the surface of plastic petri dishes arerecognized by specific antibodies in immune serum when spot-ted onto the dishes and their presence is developed with watervapor. Modifications to this procedure gave rise to the diffu-sion-in-gel ELISA (DIG-ELISA) technique (13), in which se-rum antibodies are diffused in a gel layer and their presence isdetected with a specific peroxidase conjugate anti-immuno-globulin. DIG-ELISA is a very simple assay which can becarried out with common laboratory equipment and materials,and its results can be easily read by the naked eye. DIG-ELISAhas been successfully evaluated and gave good sensitivity andspecificity values for serodiagnosis of Chagas’ disease and on-chocercosis (4, 15, 16, 23); however, results in DIG-ELISA areobtained after 30 h.

Here we report a modified version of the TIA, named theTIA-ELISA, which provides results as dots in 3 h. We com-pared it with standard ELISA, using EITB as a reference test.

MATERIALS AND METHODS

Serum samples. One hundred one serum samples previously tested in EITBwere used in a double-blind study in ELISA and TIA-ELISA, using T. spiralis E/Santigen obtained from ML. Sera were divided into two groups according to theirprevious reactivity in EITB. One group (reactive) contained 34 positive samples(8 from patients with positive muscle biopsies and 26 from patients withoutparasitoscopical studies but with symptoms compatible with trichinellosis). All ofthem showed the diagnostic bands of 44, 49, and 55 kDa and were classified inthree groups according to the intensity of reaction: weak (n 5 11), medium (n 513), and strong (n 5 10). The positive criterion is in agreement with the onedescribed before for antibody detection in swine trichinellosis (26) and in hu-mans and experimentally infected rats (8, 9). The second group (nonreactive)contained 67 serum samples from EITB-negative individuals having no symp-

* Corresponding author. Mailing address: INDRE, SSA, Carpio470, Col. Sto. Tomas, Mexico City, 11340 Mexico. Phone: (525) 3-41-49-53. Fax: (525) 3-41-32-64. E-mail: [email protected].

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toms compatible with trichinellosis. No additional data regarding donor healthwere available. All serum samples were frozen at 220°C without any preservativeadded.

Parasites. T. spiralis ML were obtained after hydrochloric pepsin (Sigma Co.,St. Louis, Mo.) digestion of muscle tissues from infected rats as previouslydescribed (9). Briefly, 4 h of digestion at 37°C was performed with constantstirring, the final suspension was filtered through three gauze layers to eliminatelarge debris, and after 45 min of sedimentation, the supernatant was discardedand the larvae were collected and washed three times in 0.01 M phosphate-buffered 0.15 M saline, pH 7.2 (PBS). Larvae were purified from the remainingfine debris through a dextrose gradient (20, 40, and 80%). Purified larvae werethen washed three times in sterile PBS and counted under the microscope.

E/S products. The E/S antigen corresponded to the supernatant of T. spiralisML (approximately 104 ML/well) cultured in 96 flat-bottom microplates filledwith RPMI 1640 medium (Gibco BRL, Grand Island, N.Y.) containing antibi-otics (penicillin G, 100 U/ml; streptomycin, 100 mg/ml) and incubated at 37°C ina humidified atmosphere containing 95% air and 5% CO2 for 48 h. Supernatantswere collected, pooled, and clarified at 750 3 g for 15 min. After proteinquantification with the Bradford dye reagent (Bio-Rad, Hercules, Calif.), aprotein inhibitor cocktail (N-tosyl-L-phenylalanine-chloromethyl ketone andtosyl-L-lysine chloromethyl ketone, 50 mg of each per ml, and phenylmethylsul-fonyl fluoride, 100 mM [final concentrations]) was added to the E/S antigen. TheE/S antigens were kept frozen at 270°C in 1-ml aliquots until used.

ELISA. ELISA was performed as previously described (8). Briefly, E/S antigen(3 mg/ml) was used to coat high-binding polystyrene plates (Costar, Cambridge,Mass.). After washing, serum samples were incubated for 120 min at 37°C.Specific antibodies were detected using a goat anti-human immunoglobulin G-phosphatase conjugate (Sigma). Substrate solution containing p-nitrophenylphosphate (Sigma) was used to develop the reaction by incubation at 37°C for 30min. The reaction was stopped by adding 1 N NaOH. Absorbance values wereobtained in an ELISA plate reader (Bio-Rad) at 405 nm. Samples were consid-ered positive when absorbance values were higher than the cutoff value estimatedas the mean plus 3 standard deviations of absorbance values from 15 negativecontrol sera that were separately studied (A405 5 0.183).

TIA-ELISA. In order to determine the optimal conditions to perform theTIA-ELISA, several concentrations of the antigen and different solutions (PBS,0.05% Tween in PBS, distilled water, 0.85% NaCl solution, and 0.1 M carbonatebuffer) to coat the plastic surfaces of petri dishes were tested using pooledpositive or negative sera. In addition, the optimal time and conditions for incu-bation of the reagents, the best dilution of serum samples and conjugate, and theusefulness of two chromogenic substances (o-phenylenediamine and 5-aminosal-icylic acid) were also determined. The best conditions identified to perform thetest were as follows. Sterile polystyrene petri dishes (Laboratorios Technicare,Mexico City, Mexico) were firmly positioned on a pattern designed to accom-modate 52 samples; the orientation was marked with a soft pen. Dishes weredotted on the marks indicated by the pattern with 10 ml of E/S antigen (7 mg/ml)diluted in 0.1 M carbonate-bicarbonate buffer (30 mM Na2CO3 and 70 mMNaHCO3, pH 9.6) and incubated for 1 h at 37°C in a wet chamber; they were thenwashed gently three times with 150 mM NaCl solution and three times withdistilled water and finally air dried. Dishes were carefully and appropriately puton the pattern, and 5 ml of a serum sample diluted 1:60 in 0.05% Tween in PBS(PBS-T) was added exactly on the site of one drop of antigen; each serum wastested on duplicate dots. Dishes were incubated for 30 min as described above,washed three times with PBS-T, then washed with PBS, air dried, and positionedagain on the pattern. Five microliters of peroxidase-conjugated goat anti-humanimmunoglobulins (Sigma), diluted 1:40 in PBS-T, was added precisely on theimmobilized immune complexes. Dishes were incubated and washed as describedabove. Agar-Agar (Dibico, Mexico City, Mexico) (0.87 g/100 ml of PBS) wasmelted and cooled to 45°C; then 10 ml of the agar solution was mixed with thesubstrate solution prepared with 5 mg of 5-aminosalicylic acid (Sigma), 1 ml ofPBS, and 20 ml of 3% H2O2. The substrate was poured onto each dish. After 30min of incubation at room temperature, the colored dots detected by the nakedeye were registered. The presence of colored reaction zones was the criterion forpositivity of the test. The intensity of reaction was arbitrarily classified as weak,

medium, and strong. Finally, dishes were covered with an appropriately sizedpiece of filter paper (Whatman no. 4), wetted in distilled water, and left un-capped on the bench for 18 h. Before the filter paper was removed, dishes wereadded and incubated with 0.5% glycerol for 5 min, dried, and stored at roomtemperature.

Serological evaluation. The serological parameters needed to perform theevaluation of TIA-ELISA, i.e., sensitivity, specificity, and predictive value for apositive and for a negative result, as well as the kappa coefficient, were deter-mined according to previously described procedures (2).

RESULTS

Antigen-antibody reactions were visualized in TIA-ELISAas brown dots formed in the gel-containing substrate (Fig. 1).As is shown, the reaction zones are almost circular and areclearly differentiated from the neighboring clear areas. Colorintensity, as well as the size and definition of the dots, is notuniform. Negative sera did not form colored spots. No changesin results were observed when complete dishes or just thegel-containing results were dried and preserved. Table 1 showsthe reactivity of TIA-ELISA and ELISA compared to EITB, aswell as the serological parameters analyzed. Sensitivity andspecificity of TIA-ELISA were similar; in contrast, ELISAshowed a lower sensitivity but a higher specificity. The predic-tive values both for positive and negative results are quitesimilar between the techniques, although for TIA-ELISA thenegative predictive value is higher. The kappa coefficient indi-

FIG. 1. Reactivity of 50 serum samples tested in TIA-ELISA against an E/ST. spiralis ML antigen. Colored dots indicate the positions of positive samples (1,3 through 7, 11 through 15, 24, 30, 35 through 39, and 43 through 47) while clearareas above the numbers indicate the positions of negative serum samples (2, 8through 10, 16 through 23, 25 through 29, 31 through 34, 40 through 42, and 48through 50). Positive and negative control serum samples are in positions 51 and52, respectively.

TABLE 1. Serological evaluation of TIA-ELISA and ELISA in immunodiagnosis of human trichinellosis

Test and result

Health condition of serum sample sourceSensitivity

(%)Specificity

(%)

Predictive value (%)Kappa coefficient

indexInfected(EITB positive, n 5 34)

Normal(EITB negative, n 5 67) Positive Negative

TIA-ELISA 91 91 84 95 0.80Positive 31 6Negative 3 61

ELISA 76 94 87 89 0.73Positive 26 4Negative 8 63

VOL. 7, 2000 TIA-ELISA IN TRICHINELLOSIS 811

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ces obtained for both tests are considered good. Compared tothe long time and high quantities of antigen and conjugateused by EITB (28 h and 2 mg of antigen and 1 ml of conjugateper sample) and ELISA (8 h and 0.63 mg of antigen and 0.1 mlof conjugate per sample), TIA-ELISA is a faster and moreeconomic test, since it requires 3 h and 0.14 mg of antigen and0.3 ml of conjugate per sample. Regarding the intensity ofreaction by TIA-ELISA, 8 samples were classified as having aweak reaction, 10 had a medium reaction, and 19 had a strongreaction. However, no association was found among tests inrelation to the intensity of the reaction (data not shown).

DISCUSSION

A novel immunoenzymatic test named TIA-ELISA was de-veloped, standardized, and evaluated for antibody detection inhuman trichinellosis by using the E/S products of T. spiralis MLas antigens. Results were compared with those obtained withthe conventional ELISA and were serologically evaluated us-ing EITB as the reference test (9, 26). During the standard-ization process it was observed that the antigen could be easilyattached to the plastic surface of the petri dish, especially whencarbonate buffer was used. Also, the enzyme-substrate reactiontook place in the dot area in the gel. Likewise, it was clearlydetermined that the peroxidase by-products remained stable inthe dried gel, provided that 5-aminosalicylic acid was used asthe chromogen as previously reported (4, 13, 15, 16).

An ideal serodiagnostic test, especially for developing coun-tries, should be cheap, simple, and easy to perform, in additionto being sensitive and specific (28). As our results show, TIA-ELISA fulfills these requirements. The test could be as usefulas EITB and ELISA for antibody detection in human trichinel-losis, since the kappa coefficient index was higher than forELISA and the sensitivity was high, meaning that TIA-ELISAcan identify more positive samples than ELISA, which is rel-evant to selecting a screening test. Furthermore, TIA-ELISA iscarried out using common laboratory equipment, and its re-sults can be read by the naked eye in no more than 3 h,although this can be further reduced to almost 2 h if petridishes are previously sensitized, because the antigenicity of theE/S antigens remains unaffected after drying. Moreover, theimmune complexes and the peroxidase activity also remainstable after each drying step; in fact, Fig. 1 is a photograph ofa dried gel in the petri dish from one experiment performed 10months ago. Due to the above-mentioned reasons, TIA-ELISAmay be a good candidate to be used in field studies, in whichgetting diagnostic information for cases of particular interestas fast as possible may be important in order to provide timelymedical attention, as required in outbreaks, or to prevent thespread of human infections. Also, TIA-ELISA is a good alter-native for clinical laboratories that lack equipment to performEITB or ELISA. Finally, TIA-ELISA could also be useful forperforming a faster diagnosis of T. spiralis infection in abattoirsbefore or immediately after slaughtering or in rurally reared orfree-roaming swine to prevent transmission to humans (5, 6,17, 24).

ACKNOWLEDGMENTS

We are thankful to S. Garcıa-Alfaro, I. Tinoco, N. Cordero, Y. Islas,and S. Zamora for excellent laboratory assistance and to D. Correaand A. Flisser for critical review of the manuscript.

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