Enzyme-Linked Immunosorbent Assay for Antibodies to

Transcription

1 INFECTION AND IMMUNITY, Feb. 1980, p /80/ /05$02.00/0 Vol. 27, No. 2 Enzyme-Linked Immunosorbent Assay for Antibodies to Toxoplasma gondii Polysaccharides in Human Toxoplasmosis JOSI, R. MINEO, MARIO E. CAMARGO, AND ANTONIO W. FERREIRA Laboratory of Immunology (Seroepidemiology), Instituto de Medicina Tropical de Sdo Paulo, University of Sdo Paulo, Sdo Paulo, Brazil A polysaccharide fraction from Toxoplasma gondii was adsorbed to polystyrene plates, and the enzyme-linked immunosorbent assay was performed (poly- ELISA) with peroxidase-labeled anti-immunoglobulin G and anti-immunoglobulin M antibodies. A comparison was made with a T. gondii total protein extract ELISA (protein ELISA) in serum samples presenting different toxoplasmosis serological patterns, as indicated by a battery of tests for toxoplasmosis. Very low titers and negative results were seen for immunoglobulin G poly-elisa both for serum samples corresponding to ancient or transitional-period infections (serological patterns II and III) and for samples of recent or acute toxoplasmosis (pattern I). On the contrary, immunoglobulin M poly-elisa furnished high titer results for pattern I sera, and a very close agreement of titers was seen between immunoglobulin M protein ELISA and immunoglobulin M poly-elisa. When the polysaccharide fraction was added to pattern I sera, a complete blocking of immunoglobulin M antibody reactivity resulted only for poly-elisa. In the same way, the total protein extract could completely block only reactivity for protein ELISA. In both cases, a limited decrease in titers was observed for respective heterologous assays. Toxoplasmosis serology is fairly complex. For clinical purposes, because of the frequent widespread occurrence of low-titer-positive sera in a population, quantitative tests are necessary to identify recent infections, which usually show high antibody titers. Tests for immunoglobulin M (IgM) antibodies are also of paramount importance for the serological diagnosis of both acute and congenital forms of the disease, as first observed by Remington et al. (8, 9). The enzyme-linked immunosorbent assay (ELISA), as well as the immunofluorescence test (IF), can be used to quantitate anti-toxoplasma antibodies and to identify such antibodies by their immunoglobulin classes. One of the advantages of ELISA is that it permits the use of defined soluble components of the parasite as antigen, whereas in the IF test a complex wholeprotein antigen must be used. In this paper we present results of an immunoenzymatic assay for antibodies to a sugar-rich macromolecular fraction of Toxoplasma gondii and compare these results with the results of a similar assay with a toxoplasma total protein extract as antigen. MATERIALS AND METHODS Serum samples. A total of 83 serum samples already tested by other toxoplasmosis serological tests and reacting in various toxoplasmosis serological patterns (3) were tested by micro-elisa, using protein and carbohydrate antigens. These samples had been stored at -20 C with glycerin (vol/vol) for periods from a few days to several months before testing. Polysaccharides from T. gondiil Parasites were obtained from the peritoneal exudates of about 100 mice, 3 days after inoculation with T. gondii, MOC strain (4). Contaminating cells were removed after agglutination with phytohemagglutinin P (Difco Laboratories, Detroit, Mich.) and filtration through nylonwool by the procedure of Hoshino-Shimizu et al. (S. Hoshino-Shimizu, J. R. Mineo, and M. E. Camargo, J. Parasitol., in press). Toxoplasmas, about 99.6% pure, were suspended in 10 ml of distilled water and submitted to ultrasonic oscillations at 40 Hz for 3 periods of 30 s each, in an ice bath. A 10-ml volume of phenol (recrystallized, saturated, and stabilized with 8-hydroxyquinoline) was added, following the procedure of Westphal et al. (10), and the mixture was kept for 10 min at room temperature under magnetic agitation. After centrifuging for 5 min at 10,000 x g, the aqueous top layer was collected and dialyzed against large volumes of distilled water for 2 or 3 days and kept as the polysaccharide fraction. No proteins could be detected in this fraction by the Lowry method (5). The polysaccharide concentration differed in different preparations, ranging from 16 to 20 jg/ml (anthrone method, with D-galactose as standard) (6). IgG and IgM micro-elisa. Immunoenzymatic assays were performed for IgG and IgM antibodies, both with a T. gondii protein extract (protein ELISA), as previously described (2), and with the polysaccharide fraction (poly-elisa). Enzymatic anti-globulin 283

2 284 MINEO, CAMARGO, AND FERREIRA conjugates were prepared by labeling anti-igg or anti- IgM antibodies with peroxidase (type VI; Sigma Chemical Co., St. Louis, Mo.). Anti-IgG serum was produced by inoculating rabbits with a myeloma IgG paraprotein, isolated by Sephadex G-200 chromatography. Anti-human IgM heavy-chain-specific globulins were commercially obtained (lot no ; Cappel Laboratories, Cochranville, Pa.). Conjugation of anti-igg and anti-igm globulins with peroxidase was done by a modified form of the technique of Nakane and Kawaoi (7; personal communication). Tests were performed as previously described (2), with 1-h incubation periods for serum, conjugate, and enzyme substrate. For substrate, 5-aminosalycilic acid was used (1), and readings were performed in 1-mm cuvettes at 450 nm in a Beckman spectrophotometer. Other tests for anti-toxoplasma antibodies. Serum samples were submitted to IF (IgG IF and IgM IF), hemagglutination, and complement-fixation tests, by techniques previously described (2). RESULTS Sensitization of plates and immunoenzymatic assays with T. gondii polysaccharide fraction. Sensitization of plates (polystyrene, Autotray, Canalco) with the polysaccharide fraction was studied by using various diluting buffers and temperatures. Best results were obtained by incubating plates overnight at 40C with the polysaccharide fraction in 0.1 M carbonate-bicarbonate buffer, ph 9.1. Figure 1 shows test results with the anti-igm conjugate, for a reactive and a nonreactive serum, in plates sensitized at different polysaccharide concentrations. For the 0.D. INFECT. IMMUN. assays, a 4-t&g/ml concentration was selected. Optical densities of and higher were considered as positive results. Each serum titer was taken as the highest serum dilution giving a positive result. Comparative results of IgG poly-elisa and IgG protein ELISA. Thirty-nine serum samples which did not contain IgM antibodies to T. gondii were tested by both immunoenzymatic assays for IgG antibodies to protein or polysaccharide antigens. These included samples reactive in toxoplasmosis tests according to patterns II or III, and nonreactive samples. Poly- ELISA gave much lower titers than protein ELISA, and even negative results for samples presenting high titers in the latter assay (Fig. 2). Similar differences were also observed for 44 serum samples (Fig. 3) from cases of acute toxoplasmosis which contained anti-t. gondii IgM antibodies and a toxoplasmosis serological pattern I. IgM poly-elisa and IgM protein ELISA in toxoplasmosis serological pattern I serum samples. Very close correlations were seen between the polysaccharide and the protein immunoenzymatic assays for IgM antibodies (Fig. 4). The same titers occurred in 20 cases, and in 90.9% of the samples (40 patients) differences were not larger than one fourfold dilution. For the remaining four sera, titer differences corresponded to two serum dilutions. Blocking of ELISA serum reactivity by toxoplasma components. Toxoplasma poly- 1/16 1/64 1/256 1/1024 1/400 SERUM DILUTIONS FIG. 1. IgM ELISA with different sensitizing concentrations of the polysaccharide fraction. (-----), Results for a negative serum with sensitizing antigen at 8 pg/ml.

3 VOL. 27, 1980 saccharide fraction or total protein extract was added to samples presenting serological pattern I and reactive in both IgM immunoenzymatic assays. Final concentrations were, respectively, 5 yg/ml for polysaccharide and 10,ug/ml for total protein extract, in a 1:16 serum dilution. After incubation at 4VC overnight and for 1 h at 370C, the mixtures were further diluted and assayed. Complete blocking of homologous ELISA was obtained in either case, with only a small titer reduction in the heterologous assay. DISCUSSION For clinical purposes, identification of T. gondii serum antibodies by IgM and IgG classes has been of much value in the diagnosis of both recent infections and congenital forms of the disease. Identification of antibodies by specificity for different parasite components may also acquire diagnostic importance, as suggested by a few clues. For example, surprising changes are 10G-protpM ELISA 64,000 3cooq 16,00o 6,00o0 4,00OF i,oo4 2"[ POLYSACCHARIDE ELISA: TOXOPLASMOSIS SEROLOGY *@/ 0S 6 0. *@ 00~ ~~0 * frequently observed for hemagglutination test titers of IgG antibodies in the course of the infection. Thus, a sudden rise of previously low hemagglutination titers to the already high IgG IF titers represents a serological mark between acute toxoplasmosis pattern I and transition pattern II as constant as the reversion to negative of the IgM IF test (3). As a first step in the study of immunologically reactive parasite components, a polysaccharide fraction was isolated from a pure toxoplasma suspension. Although this fraction could not be characterized as a glycoprotein since no protein components were found, the presence of nonaromatic amino acids could not be dismissed. We could better characterize this fraction as a sugar-rich macromolecular component. Immunoenzymatic assay of antibodies to this fraction was made possible since it could be adsorbed to polystyrene plates. A maximal sensitization of the plastic surface was reached at a 4-,tg/ml polysaccharide concentration..4 l1 >li 0 * I 1 ol a I I > ,000 4,000 6,000 16,000 32,000 64,000 IgG -poly ELISA FIG. 2. Comparative results of IgG poly-elisa and IgG protein ELISA in 39 serum samples from toxoplasmosis serological patterns II and III, or from nonreactive samples.

4 IgG - protein ELISA 64,000 * * * 32, ,000 / 8,000 - ~ ~ ~ 00 6,000 7 ** 4,000 1,000- *. ~ ~ ~ ~ 0 s*35 ISS -~~~~~~,o66./ >16 > t26 1,000 4,000,000 16,000 32,000 64,000 IG -poly ELISA FIG. 3. Comparative results of IgG poly-elisa and IgG protein toxoplasmosis serological pattern I (acute toxoplasmosis). ELISA in 44 serum samples from 1gM -protein ELI SA 16,000, 6, , I I ,000 4,000 6,000 16O0 32,000 1gM -poly ELISA FIG. 4. Comparative results ofigmpoly-elisa and IgMprotein ELISA in 44 samples from toxoplasmosis serological pattern I (acute toxoplasmosis). 286

5 VOL. 27, 1980 POLYSACCHARIDE ELISA: TOXOPLASMOSIS SEROLOGY 287 For serum samples presenting an old infection (serological pattern III), no IgM antibodies for the polysaccharide fraction were found. Also, anti-polysaccharide IgG antibodies were only occasionally observed, and in low titers when present. This was the case even when very hightiter anti-protein IgG antibodies were found. For pattern I serum samples from recent or acute infections, anti-polysaccharide IgM antibodies were constantly observed. A strict correlation was seen between IgM antibody titers in both poly-elisa and protein ELISA. However, anti-polysaccharide IgG antibodies were observed only occasionally and in low titers for acute pattern sera. When mixing either the total extract or the polysaccharide fraction with an IgM ELISA-reactive serum, negative reactions in the homologous assays were observed, whereas for the heterologous ones only a small decrease in titer resulted. In human toxoplasmosis, it seems that initially a strong IgM antibody response frequently occurs to determinants both in the sugar and protein moieties of a glycoprotein complex. However, the IgG antibody response to polysaccharides is always low or even absent, but is marked against parasite protein components. LITERATURE CITED 1. Bullock, S. L., and K. W. Walls Evaluation of some of the parameters of the enzyme-linked immunospecific assay. J. Infect. Dis. 136: Camargo, M. E., A. W. Ferreira, J. R. Mineo, C. K. Takiguti, and 0. S. Nakahara Immunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns. Infect. Immun. 21: Camargo, M. E., and P. G. Leser Diagnostic information from serological tests in human toxoplasmosis. II. Evolutive study of antibodies and serological patterns in acquired toxoplasmosis, as detected by hemagglutination, complement fixation, IgG- and IgMimmunofluorescence tests. Rev. Inst. Med. Trop. S. Paulo 18: Jamra, L. M. F Contribuigio para a epidemiologia da toxoplasmose. Inquerito em 100 familias de uma area da Cidade de Sio Paulo. Tese apresentada a Cadeira de Doengas Tropicais e Infecciosas da Faculdade de Medicina da Universidade de Sao Paulo. Sao Paulo, Brazil. 5. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Martirani, I., G. Hoxter, B. L. Waschenberg, L. Mariani, and A. B. U. Cintra Determination of polysaccharide hexoses and hexosamines in normal human sera. J. Lab. Clin. Med. 54: Nakane, P. K., and A. Kawaoi Peroxidase-labelled antibody. A new method of conjugation. J. Histochem. Cytochem. 22: Remington, J. S., M. J. Miller, and L. Browlee IgM antibodies in acute toxoplasmosis. I. Diagnostic significance in congenital cases and a method for their rapid demonstration. Pediatrics 41: Remington, J. S., M. J. Miller, and I. Browlee IgM antibodies in acute toxoplasmosis. II. Prevalence and significance in acquired cases. J. Lab. Clin. Med. 71: Westphal, O., O. Luderitz, and L. Bister Uberdie extraction von Bakterium mit phenol/wasser. Z. Naturforsch. 76: