Toxoplasma gondii Which Is Not Affected by Rheumatoid Factor or Immunoglobulin G Antibodies

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1 JOURNAL OF CLNCAL MCROBOLOGY, Jan. 1986, p /86/177-6$2./ Copyright 1986, American Society for Microbiology Vol. 23, No. 1 An Enzyme mmunoassay for mmunoglobulin M Antibodies to Toxoplasma gondii Which s Not Affected by Rheumatoid Factor or mmunoglobulin G Antibodies TSUE-MNG LN,'* MONROE W. CHN-SEE,' SEYMOUR P. HALBERT,' AND J. MEHSEN JOSEPH2 Cordis Laboratories, Miami, Florida 33127,' and Laboratories Administration, Department of Health and Mental Hygiene, State of Maryland, Baltimore, Maryland Received 2 July 1985/Accepted 28 September 1985 An enzyme-linked immunosorbent assay (ELSA) for total antibodies to Toxoplasma gondii was modified to measure specific immunoglobulin M (1gM) antibodies. The assay requires three incubation periods totaling 2 h and enzyme-labeled heavy-chain-specific antibodies to human gm. The objective read-out in absorbance was normalized to percent of a standardized positive control for interpretations. No difference was observed between the assay results with or without previous absorption of the samples by Staphylococcus aureus protein A to remove most of the gg antibodies. Addition of serum containing very high levels of gg antibodies to another containing both gg and gm antibodies did not change the gm assay values for the latter. None of the 22 sera containing high levels of gm rheumatoid factor (RF) gave positive ELSA gm results, even though 8 of them also had high levels of gg toxoplasma antibodies. Mixtures of sera containing high concentrations of RF with sera having high levels of gg toxoplasma antibodies also failed to show any false-positive reactions in the gm toxoplasma assay. Thus, this ELSA for T. gondii gm antibodies was not affected by gg toxoplasma antibodies and RF. Serological tests for antibodies to Toxoplasma gondii are often helpful for the diagnosis of active infection by this organism as well as for indicating previous exposure (12, 17, 25, 27). Of the assays most widely available, indirect immunofluorescence (F) and enzyme-linked immunosorbent assay (ELSA) readily permit the detection of different antibody classes (1, 6, 11, 25, 26). Although the presence of very high levels of total or immunoglobulin G (gg) antibodies is indicative of active disease (17), the detection of gm antibodies suggests an acute current infection, since the antibody response of this immunoglobulin class tends to be transient and is not usually associated with chronic or recurring disease (4, 24, 28). A number of studies concerned with the detection of gm antibodies to various infectious agents showed that competing gg antibodies or gm rheumatoid factor or both must be removed to eliminate errors in the results (2, 3, 5, 1, 16, 21). Our group has previously described a standardized ELSA procedure for measuring total antibodies to Toxoplasma gondii (13). This method has now been modified by use of the appropriate conjugate to measure only gm antibodies to this organism. Unexpectedly, the new version of the assay was shown to be unaffected by high levels of gm rheumatoid factor (RF) or gg antibodies (or both) to this organism, as described in the present report. MATERALS AND METHODS Test serum samples. Normal serum samples were obtained from 223 apparently healthy adult volunteers working in four different facilities in Miami. Sera from 12 patients suspected of having active toxoplasma infections, based on gm immunofluorescence assays results or clinical evaluation or both, were kindly made available to us by seven research groups from various parts of the world. We are deeply grateful to the following for these specimens: M. E. Camargo, * Corresponding author. 77 nstituto de Medicina Tropical de Sao Paulo, Brazil; E. K. Petterson, Food and Agricultural Organization/World Health Organization Collaborating Center for Research and Reference on Toxoplasmosis, Copenhagen, Denmark; J. E. Organ, Alabama State Health Department Laboratory, Montgomery, Ala.;. Lebrun, Laboratories Merck-Clevenot, Chelles, France; M. A. Gordon, New York State Health Department, Albany, N.Y.; K. W. Walls, Centers for Disease Control, Atlanta, Ga.; and Dr. Roy Stevens, New York State Health Department, Albany, N.Y. n addition, quantitative immunofluorescence assays were carried out at Cordis Laboratories with 14 of these sera from patients and normal individuals by using test reagents from Microbiological Research, Bountiful, Utah. gm rheumatoid factor was assayed by CORDA RF (Cordis, Miami, Fla.) (7). gg antibodies to T. gondii were measured by the ELSA described previously but with the use of heavy-chain-specific enzyme-labeled antibodies to human gg and a standard calibrated from the 1959 World Health Organization international standard for anti-toxoplasma serum (13). All serum samples were stored at -2 C. ELSA for gm antibody to T. gondii (ELSA-gM). The reagents and assay procedure were the same as described previously for total antibody (13), with two major modifications. The enzyme-labeled antibody was highly specific for human gm heavy chains and the test results were reported as the percentage of positive control absorbance for interpretation. Briefly, Toxoplasma organisms (Rh strain) were harvested from the peritoneal cavity of 3-day-infected mice, washed four times with cold saline, and frozen at -2 C. They were thawed, sonicated, and cleared at high speed (45, x g, 3 min) to obtain a clear antigen solution which was used to coat special plastic disks containing isothiocyanate groups. For the assay, 1,ul of test sera, as well as positive and negative control sera, were diluted with 5,ul of 6% bovine serum albumin specimen diluent and incubated under constant shaking with a T. gondii antigen-coated disk Downloaded from on October 1, 218 by guest

2 78 LN ET AL. at 37 C for 45 min. The disks were thoroughly washed with Tris-saline, transferred to clean vials, and incubated with 5 p1l of enzyme-labeled antibody to human gm at 37 C as above for 45 min. The disks were washed, transferred to clean vials, and incubated 3 min with 1 ml of p-nitrophenyl phosphate substrate solution. The reaction was stopped with.1 ml of 3 M NaOH and read at 45 nm. The absorbance readings of test samples were divided by that of the standardized positive control to obtain the percentage of positive control value. A pool of two Toxoplasma gm-positive sera was used to prepare the primary reference standard, against which all positive controls for the ELSA-gM test were calibrated. Numerous samples of this primary standard were stored at -2 C for calibration of additional lots of positive control. The positive sera used were positive for gm Toxoplasma antibodies by both ELSA and F. The positive sera used for the standard and for positive controls in the assay were also confirmed to contain gm antibodies to T. gondii by ELSA-gM and F-gM assays of their isolated gm fractions (obtained from the void volume of a Sephadex G-2 gel filtration, i.e., molecular weight > 2,). Rheumatoid factor was absent from the primary standard and each of the positive controls. Reproducibility of test results with six serum samples covering a wide range of activities (8 replicates) revealed an average coefficient of variation of 9.2%. The average run-torun coefficient of variation for 11 serum samples was 7.3%. The immunological specificity of the ELSA gm for T. gondii was confirmed by blocking experiments with a variety of antigens as described previously (13) and by negative test results with two macroglobulinemia sera containing extremely high gm concentrations (1,65 and 2,4 mg/1 ml, normal 39 to 117 mg/1 ml). Absorption of gg in test sera with protein A-containing Staphylococcus aureus. The Cowan strain of S. alireias was obtained from the American Type Culture Collection (ATCC-3). After growth, 1% killed organism suspensions (Formalin and heat) were prepared as described by Kessler (9). For use, 1 pl. of test serum sample was added to 1 pl. of this bacterial suspension which had been diluted with 4 pl. of 6% bovine serum albumin specimen diluent, and the mixture was preincubated for 45 min at 37 C with shaking. A toxoplasma antigen-coated disk was then added to initiate and complete the ELSA-gM procedure. RESULTS The cut-off points for the ELSA-gM were determined with data obtained from assaying serum samples of 223 healthy normal volunteers and 84 patients. The mean ELSA-gM value (percentage of positive control) for the 223 normal subjects was 68, with a standard deviation of 26. The mean + 2 standard deviations = 12. The distribution of the ELSA-gM values of these 223 normal and 84 patient sera is shown in Fig. 1 and revealed a demarcation of normal and patient ELSA-gM levels at 1% positive control. t was therefore established that the specimens with an ELSA-gM value of 12 and greater should be considered positive for gm antibodies to T. gondii, 1 to less than 12 were equivocal, and less than 1 were negative. On the basis of these criteria, of the 223 normal sera, 25 (92%) were negative, 11 (4.9%) were equivocal, and 7 (3.1%) were positive. t is of interest that most of these positive (5 of 7) and equivocal (8 of 11) sera were from individuals working in the same building. Of the 84 patient sera, 57 (68%) were positive, 12 (2%) were equivocal, and 1 (12%) were negative. Excluding those samples with equivocal results, the predictive values of a positive and a negative were 89 and 95%, respectively; the sensitivity and specificity were 85 and 97%, respectively. A number of the patient sera with equivocal or negative ELSA results had been found by the original investigators to be positive by their F-gM procedure with whole serum, but to be negative with the isolated gm fraction (three specimens), or vice versa (six specimens). n separate evaluations, a total of 52 serum samples were assayed by this ELSA-gM at two public health laboratories in comparison with their own F-gM procedures. The results showed good agreement between the ELSA-gM and their F-gM results (Fig. 2), confirming the cut-off points for the ELSA-gM. The international standards for anti-toxoplasma sera 1959 and 198 prepared by the World Health Organization, which were weakly positive by the F-gM (World Health Organization Expert Committee on Biological Standardization, April 198, Geneva), also gave weakly positive values of 13 and 133, respectively, by this ELSA-gM. Correlation between the ELSA-gM values and F-gM titers. The ELSA-gM values were compared to the quantitative F-gM titers of specimens received from seven of the eight laboratories, involving 7 to 39 specimens in each group. The eighth laboratory submitted only three specimens, and their data could not be analyzed separately. The data from each laboratory were not pooled for analysis because of the considerable technical differences in the 3 w- 5' () a- o2 12 _ 1. _ 1 J. CLN. MCROBOL. V a POSVE EQUVOCAL NEGATVE HEALTHY PATENTS VOLUNTEERS F-GM POS. (N-223) (N(84) FG. 1. The ELSA-gM values of serum samples from 223 normal healthy volunteers and from 84 patients reported positive for gm antibodies to T. gondii by F-gM. Downloaded from on October 1, 218 by guest

3 VOL. 23, 1986 procedures and reagents used by these various laboratories, resulting in widely different cut-off titers for normal (undiluted to 1:4). The ELSA-gM yalues showed significant correlation with F-gM titers (P <.1) in each case. Two of these correlations are illustrated in Fig. 3, revealing correlation coefficients of.87 and.82. p addition, 84 sera from patients and 2 from healthy volunteers were coded and assayed at Cordis Laboratories with a commercial, F-gM kit after fou'rfold serial dilutions (1:4 to 1:1,24). The results revealed a significant correlation between the F-gM titers and ELSA-gM values for these 14 serum samples, with a correlation coefficient of.75 (P <.1). The correlation coefficient between the F-gM titers and the median ELSA-gM values at each of the five F titer groups (negative, 16, 64, 256, and 1,24) was.982 (P <.1) (data not shown). gg antibodies in the test samples and their removal by absorption with staphylococcal protein A. The 84 patient sera analyzed had all be'en found by the original investigators to contain both gm and gg antibodies with F procedures. These patient sera, 98 normal sera, and the positive control were absorbed with S. aureus before assay by ELSA-gM to determine whether removal of gg antibodies would appreciably change the ELSA-gM values. No significant differences in the ELSA-gM values were seen between the assay results with and without prior S. aureus absorption 4- o3- U. 1- of E 4 co 1. i T POS EQ * NEG A ELSA FOR gm ANTBODES TO T. GOND 79 a. 2-4Co w 21 O R-O.7 *1 \6 4 \, F-GM TTER R-.82 s/.t w1^4\9t FG. 3. Scatter diagrams showing correlations between toe ELSA-gM values and the F-gM titers for T. gondii reported by source investigators. R, Correlation coefficient. (Fig. 4). There was a small parallel reduction in absorbance value for all sera and controls after the absorption. Some of these serum samples had considerably higher F-gG titers than F-gM titers, and still others showed HF- gm-positive reactions oniy with the isolated gm fractions, yet removal of most gg by S. aureus did not significantly change the ELSA-gM results. As controls to document the gg absorption, all 98 normal as well as 11 patient sera were also assayed for gg antil4odr ies to T. gondii with or without prior S. aureus absorption. One third of the normal sera were positive for gg antibodies (3 to greater than 5 U/ml), but their negative ELSA-gM values showed the same distribution as those of gg antibody-negative sera. The S. aureus pretreatment removed more than 9% of gg antibodies and converted most of the gg antibody-positive (some being very strong) sera to *- ii z~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~ WVA - 1 ~~~~~~*,~~~~~~ * D Downloaded from on October 1, 218 by guest - i4 LABORATORY A LABORATORY B FG. 2. Evaluations of the ELSA-gM at two public health laboratories. One test sample (A) was negative when tested with its whole serum but positive with its gm fraction by their F-gM assay, while another (V) was vice versa. z ELSA-GCKOPLASA, % OF POSTVE CONTROL FG. 4. A scatter diagram showing the ELSA-gM values of 98 normal () and 84 patient () sera with and without absorption b,y S. aureus before assay. The line is at 45, indicating equivalence between results with and without absorption.

4 8 LN ET AL. J. CLN. MCROBOL. W -J.D U) 45 wcr 'i uii P. im (D z (D wil 11- ::... ~~~~~ ~ 5.?... _o K 3~5 ELSA-GG-TOXOPLASMA, V/ML FG. 5. gg antibodies to T. gondii in 98 normal () and 11 patient () sera assayed by the ELSA procedure with and without a prior absorption by S. qulreus. TABLE 1. Absorption by S. auireius of gg and gm antibodies to T. gondii in sera from 11 patients ELSA results Original reported Sample gm antibodies (% gg antibodies F titer P positive control)" (lu/ml) Untreated Absorbed Untreated Absorbed gg gm 1' ' ' ' d >543 >772 > , ,192 1, >1, 16 6, , ,384 ' ND' ND a b <1, negative;.12, positive; -1 but <12, equivocal. By ELSA gg; <3 negative. ' Contained RF (57, 76, 126, and 82 lu/ml); RF after S. aireis absorption, 3, 4, 26, and 2 lu/ml, respectively, for samples 1 to 4. Negative, <1 lu/m. d Negative when gm fraction was assayed. e Positive (16) when gm fraction was assayed. fnd, Not done. also fail to reveal any evidence of interference by gg antibodies in the ELSA-gM. RF in the test sera. Eighteen RF-positive sera were assayed for toxoplasma gm antibodies by ELSA. Although most of them, were extremely strongly positive for RF (normal, <1 U/m), all were found to be negative for gm toxoplasma antibodies (Fig. 7). Three of these RF-positive sera were strongly and one was weakly positive or gg antibodies tq T. gondii, an incidence rate found in the normal population (25). Sera from four patients supplied by other investigators were considered to be false positive by lf-gm because they contained both RF and gg antibodies to T. gondii and their gm fractions were negative by TF'. Assays by our ELSA procedures confirmed that they all had moderate to high levels of grl antibodies to T. gondii (135 to 498 U/ml) as well as RF (57 to 126 U/ml). However, they'all failed to show positive results in the ELSA-gM assay (Table 1 and Fig. 7). Absorption of these sera with S. aurei, dramatically reduced their gg antibodies and RF (Table 1), but the negative reactions, while their ELSA-gM values remained 1ELSA-gM values were not appreciably altered, showing about the same (Fig. and Table 1). These results indicate no effect by gg antibodies and RF. that the ELSA-gM reactivities are unaffected by various Four serum samples containing high levels of toxoplasma levels of gg toxoplasma antibodies in individual sera. gg antibodies wdere diluted with an equal volume of two This finding was further supported by titration experiments of gm-positive serum in diluents which did or did not 592 U/ml; normal, <1). Three of the four test sera con' serum samples containi'ng very high levels of RF (64 and contain high levels of gg toxoplasma antibodies. A test tained no toxoplasma gm antibodies, while the fourth was serum containing high concentrations of toxoplasma gg and weakly positive only when assayed undiluted. A fifth seru.m gm antibodies (F titers 1:16,384 and 1,24, respectively) which was negative for both RF and antibodies to T. gondii was serially diluted fourfold (1:16 to 1:1,24) with: (i) served a as a control. The mixtures were assayed by ELSAserum with a high F-gG titer (1,24) but negative by gm, and the results (Table 2) revealed that addition of high F-gM; (ii) a serum negative for both gg and gm antibodies as a control; and (iii) bovine serum albumin specimen did not cause false-positive reactions to occur in the ELSA- concentrations of RF ipto gg antibody-containing serum diluent, following which each 1,ul of dilution received 1,ul 1gM. The same serum mixtures were also absorbed with of undiluted serum containing strong gg but no gm antibodies. The three serial dilutions were assayed by ELSA- found above in Fig. 4, the gm toxoprasma antibody values protein A S. aureus suspensions before ELSA-gM. As gm, and the results were, plotted against the extrapolated were not significantly affected by this pretreatment which lf-gm titer (Fig. O). The titration curves of dilutions in the removed gg, but the RF activ,ity was decreased at least 95% two sera containing gg antibodies were not significantly by this absorption step. different from those in their absence. These observations 5.5 jd5# AV l GM GG EXTRAPOLATED llf TR OF DLUrON N/WTH GG ANT1BOD' SWUM FG. 6. ELSA-gM results of serial dilutions of a.serum containing both gg and gm antibodies to T. gondii (lp' titers 16,384 and 1,24, respectively). t was serially diluted in a serum containing gg but not gm antibodies (F-gG, titer 1,24) (A) or ir) specimen diluent which had been spiked with the same amount (1,ul) of a serum containing gg but not gm antibodies (V); as controls, a serum sample lacking antibody to T. gondii () was substituted. Downloaded from on October 1, 218 by guest

5 DSCUSON Solid-phase serdlogical assays for specific antibodies of the gm class have often been reported to be unfavorably influenced by two principal factors (2, 3, 8, 1, 21). The high levels of gg antibodies present are felt to compete for-the antigens involved, resulting in false negative or appreciably reduced reactions. On the other hand, gm RF is thought to cause falsely positive reactions, since RF could react secondarily with gg which is deposited on the primary antigen in the system. For reasons that are not clear, neither of these disturbing effects was encountered in the present assay for gm antibodies to T. gondii. With regard to the competing gg antibodies, the present study revealed that with the solid phase antigen used: (i) widely varying levels of toxoplasma gg antibodies were not related to ELSA-gM values in different sera; (ii) substantial removal of gg antibodies by protein A did not sigriificantly affect the observed ELSA- gm values; (iii) deliberate addition of high concentrations of toxoplasma gg antibodies to sera containing gm antibodies did not affect the ELSA-gM values. t is possible that the present ELSA-gM procedure employed sufficient quantities of adequately purified antigens covalently bound to the solid phase so that both gm and gg antibodies present could be satisfactorily accommodated. t is possible that certain of these antigens reacted more strongly only with gm antibodies (15, 19). t is also conceivable that incubation at 37 C favored the gm antibody reactions, perhaps similar to long incubations in other immune systems which have been reported to favor gm reactivity (3, 22, 23). n this connection, Camargo et al. (1) have found that ELSA was more sensitive than F for detecting toxoplasma gm antibodies, while both tests were equally sensitive for determining gg antibodies. The false-positive gm reactions due to RF in such tests ui R 6 ao i (D 9 w VOL. 23, ioo-m. 1 POS 13L MO zoo 13 loll 113l SO RHElMATOD FAC O, UNL FG. 7. ELSA-gM results of sera containing RF. Symbols:, from one of the investigators, with additional dhta in Table 1;, from our RF serum panel. Eight specimens were positive for gg antibodies to T. gondii by the ELSA procedure and their values in international units per milliliter are marked to the left of the appropriate point. RF values of <1 U/ml are negative. ELSA FOR 1gM ANTBODES TO T. GOND 81 TABLE 2. Lack of effect of RF on toxoplasma 1gM ELSA" ggb ELSA gm' assay results of test serum Test antibodies plus equal volume of: serum to T. gondii Normal RF-positive RF-positive (lu/mi) serumd serum Ae serum B" atest serum samples lacking toxbp)asma gm antibodies but containing high titers of gg antibodies were mix,ed with equal volumes of two strong RF sera. They were then assayed for gm toxoplasma antibodies by ELSA. For controls, the same test sera were mixed with normal human serum lacking any toxoplasma antibodies or RF. n addition, a toxoplasma antibody-negative serum was also mixed with the RF-positive sera and assayed for toxoplasma gm by ELSA. n test sera by ELSA gg; <3 negative. ELSA gm, <1, negative; -12, positive;.1 but <12, equivocal. d Normal serum negative for toxoplasma antibodies and RF. erf concentrations determined by CORDA RF were: A, 64 U/ml; B, 584 U/ml (normal serum, '1 U/mi). have been documented for gm antibodies to T. gondii by specific immunofluorescence with whole organisms as antigens (2, 8). This difficulty has also been encountered in developing a similar assay in our own laboratory for cytomegalovirus antibodies of the gm class, also employing whole virus as antigens (D. Kiefer, personal communication). t is not understood why this problem was not encountered in the present assay for detecting gm toxoplasma antibodies, using soluble antigens on a solid phase. However, it was clear that specimens containing extremely high levels of both gm RF and gg antibodies to toxoplasma did not yield false-positive reactions in this assay. Furthermore, addition of large amounts of RF to sera containing high concentrations of gg toxoplasma antibodies did not create this problem. Absorption of the latter mixtures with S. aureus protein A did not affect the low gm toxoplasma reactivities, but they drastically reduced the gg toxoplasma antibody concentrations as well as the RF activities. t is of interest to note that Middledorp et al. reported that sera containing both gg antibodies to cytomegalovirus and RF did not give false-positive reactions in their microtiter ELSA for gm antibodies to cytomegalovirus (14). Partanen et al. also showed that, in the immunoblotting ELSA test for rubella gm antibodies, purified RF added to ggpositive, gm-negative serum did not give false-positive reactivity (18). Pettersen found that "only a small fraction of the false-negative results due to high gg titers and falsepdsitive results due to RF, experienced in the indirect fluorescent antibody test, were apparent in (a commercial ELSA) gm kit," using soluble antigens coated on plastic tubes (2). None of these reports gave any explanation as to why gg antibodies or RF or both show much less or no interference on gm antibody assay in the ELSA procedures. This phenomenon is worth further study and may conceivably be related to the physical state of the antigen usdd, i.e., soluble or particulate. Of the four groups of normal healthy volunteers studied in the present investigation, each worked in a different building. The incidence of gm toxoplasma positive results in one group was much higher than in thd othe&s (6 versus 1%). This group also showed a similarly high incidence of equivocal reactors. The basis for this is also not clear and is being investigated. 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6 82 LN ET AL. LTERATURE CTED 1. Camargo, M. E., A. W. Ferreira, J. R. Mineo, C. K. Takiguti, and. S. Nakahara mmunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns. nfect. mmun. 21: Camargo, M. E., P. G. Leser, and A. Rocca Rheumatoid factors as a cause for false positive gm anti-toxoplasma fluorescent tests. A technique for specific results. Rev. nst. Med. Trop. Sao Paulo 14: Cohen,. R., L. C. Norins, and A. J. Julian Competition between and effectiveness of gg and gm antibodies in indirect fluorescent antibody and other tests. J. mmunol. 98: Desmonts, G., Y. Naot, and J. S. Remington mmunoglobulin M-immunosorbent agglutination assay for diagnosis of infectious diseases: diagnosis of acute congenital and acquired toxoplasma infections. J. Clin. Microbiol. 14: Filice, G. A., A. S. Yeager, and J. S. Remington Diagnostic significance of immunoglobulin M antibodies to Toxoplasma gondii detected after separation of immunoglobulin M from immunoglobulin G antibodies. J. Clin. Microbiol. 12: Franco, E. L., K. W. Walls, and A. J. Sulzer Reverse enzyme immunoassay for detection of specific anti-toxoplasma immunoglobulin M antibodies. J. Clin. Microbiol. 13: Halbert, S. P., J. Karsh, and M. Anken A quantitative enzyme immutoassay for gm rheumatoid factor using human immunoglobulin G as substrate. Am. J. Clin. Pathol. 74: Hyde, B., E. V. Barnett, and J. S. Remington Method for differentiation of non-specific from specific toxoplasma 1gM fluorescent antibodies in patients with rheumatoid factors. Proc. Soc. Exp. Biol. Med. 149: Kessler, S. 'W Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. mmunol. 115: Knez, V,, J. A. Stewart, and D. W. Ziegler Cytoriegalovirus-specific gm and gg response in humans studied by radioimmunoassay. J. mmunol. 117: Konishi, E., and J. Takahashi Reproducible enzymelinked immuinosorbent assay with a magnetic processing system for diagnosis of toxoplasmosis. J. Clin. Microbiol. 17: Krahenbuhl, J. L., and J. S. Remington The immunology of Toxoplasma and toxoplasmosis, p n S. Cohen and K. Warren (ed.), mmunology of parasitic infections, 2nd ed. Blackwell Scientific Publications, London. 13. Lin, T-M., S. P. Halbert, and G. R. O'Connor Standardized quantitative enzyme-linked immunoassay for antibodies to Toxoplasma gondii. J. Clin. Microbiol. 11: Middledorp, J. M., J. Jongsma, A. Haar, J. Schirm, and T. H. The Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigens by enzymelinked immunosorbent assay. J. Clin. Microbiol. 2: Mineo, J. R., M. E. Camargo, and A. W. Ferreira J. CLN. MCROBOL. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polysaccharides in human toxoplasmosis. nfect. mmun. 27: Ordonez, G. A., J. T. Newman, and M. J. Stone Serological diagnosis of Toxoplasma gondii infections by rapid separation of serum immunoglobulins M and G with CM Bio-Gel A. J. Clin. Microbiol. 16: Palmer, D. F., J. J. Cavallaro, K. Walls, A. Sulzer, and M. Wilson A procedure guide to the performance of the serology of toxoplasmosis. Centers for Disease Control, Atlanta, Ga. 18. Partanen, P., H. Seppanen, J. Suni, and A. Vaheri Selective reactivity of antibodies to human immunoglobulin G, M, and A with rubella virus proteins. J. Clin. Microbiol. 21: Partanen, P., H. J. Turunen, R. Paasivuo, E. Forsblom, J. Suni, and P.. Leinikki dentification of antigenic components of Toxoplasma gondii by an immunoblotting technique. FEBS Lett. 158: Pettersen, E. K nvestigation of two commercially available kits for determination of gg and gm to Toxoplasma gondii by enzyme-linked immunosorbent assay (ELSA). Acta Pathol. Microbiol. mmunol. Scand. Sect. C 92: Pyndiah, N., U. Krech, P. Price, and J. Wilhelm Simplified chromatographic separation of immunoglobulin M from G and its application to Toxoplasma indirect immunofluorescence. J. Clin. Microbiol. 9: Schmitz, H., and M. Schere gm antibodies to Epstein- Barr virus in infectious mononucleosis. Arch. Gesamte Virusforsch. 37: Skaug, K., and E. T7jotta Diagnosis of recent herpes simplex infections. Acta Pathol. Microbiol. Scand. Sect. B 82: van Loon, A. M., J. T. M. van der Logt, F. W. A. Heesen, and J. van der Veen Enzyme-linked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay. J. Clin. Microbiol. 17: Walls, K. W Serodiagnosis of toxoplasmosis. Lab. Manag. 16: Walls, K. W., S. L. Bullock, and b. K. English Use of the enzyme-linked immunosorbent assay (ELSA) and its microadaptation for the serodiagnosis of toxoplasmosis. J. Clin. Microbiol. 5: Welch, P. C., H. Masur, T. C. Jones, and J. S. Remington Serologic diagnosis of acute lymphadenopathic toxoplasmosis. J. nfect. Dis. 142: Wielaard, F., H. van Gruijthuijsen, W. Duermeyer, A. W. L. Joss, L. Skinner, H. Williams, and E. H. van Elven Diagnosis of acute toxoplasmosis by an enzyme immunoassay for specific immunoglobulin M antibodies. J. Clin. Microbiol. 17: Downloaded from on October 1, 218 by guest