Immunoglobulin M from Immunoglobulin G Antibodies

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1980, p. 336-342 0095-1 137/80/09-0336/07$02.00/0 Vol. 12, No. 3 Diagnostic Significance of Immunoglobulin M Antibodies to Toxoplasma gondii Detected After Separation of Immunoglobulin M from Immunoglobulin G Antibodies GREGORY A. FILICE,2 ANNE S. YEAGER,' AND JACKS.REMINGTON2* Division of Infectious Diseases, Department of Medicine,' and Division of Infectious Diseases, Department of Pediatrics,3 Stanford University School of Medicine, Stanford, California 94305, and Division ofallergy, Immunology and Infectious Diseases, Palo Alto Medical Research Foundation, Palo Alto, California 943012 Failure to demonstrate immunoglobulin M (IgM) antibodies by indirect immunofluorescence (IgM-IFA) in sera from some patients with acute acquired toxoplasmosis has recently been attributed to an inhibitory effect of high titers of IgG antibodies in these sera (Pyndiah et al., J. Clin. Microbiol. 9:170-174, 1979). To confirm these findings and define their importance for diagnosis, we used gel filtration to separate IgM from IgG antibodies in a series of sera that were negative in the IgM-IFA test. A total of 68 sera were from patients with acquired toxoplasmosis, 13 were from uninfected adults, 13 were from infants with congenital toxoplasmosis, and 7 were from uninfected neonates. Of the 68 sera from patients with acquired toxoplasmosis, IgM preparations (from the separated sera) were positive in the IgM-IFA test in 36 (53%). There was a significant (P = 0.00003) association between high titers of IgM-IFA antibodies in the IgM preparations (corrected for dilution of IgM antibodies by the gel filtration procedure) and recent acquisition of infection. IgM antibodies were also detected in 5 (38%) of the IgM preparations of 13 sera from congenitally infected infants but not in any of the IgM preparations of sera from uninfected neonates. IgG antibodies to Toxoplasma gondii were shown to interfere with demonstration of IgM antibodies in the IgM-IFA test. Treatment of sera with protein A resulted in greater dilution of IgM antibodies and less efficient separation of IgM from IgG antibodies than did separation of sera by gel filtration. Treatment of sera with protein A did not result in increased detection of IgM antibodies to T. gondii. Testing of IgM preparations (obtained by gel filtration) resulted in a significant increase in sensitivity of the IgM-IFA test for the diagnosis of recently acquired and congenital toxoplasmosis. Detection of immunoglobulin M (IgM) antibodies to Toxoplasma gondii by indirect immunofluorescence (IgM-IFA) (13) is useful for the diagnosis of acute acquired toxoplasmosis (6, 14; P. C. Welch, H. Masur, T. C. Jones, and J. S. Remington, J. Infect. Dis., in press) and congenital toxoplasmosis (12). However, negative results are obtained in some sera from patients with acute acquired toxoplasmosis (7, 16) and in sera from approximately 25 to 50% of infants with congenital toxoplasmosis (12). In patients with acute acquired toxoplasmosis, negative results are usually due to failure to obtain serum early in the illness. In some infants with congenital toxoplasmosis, negative results are due to lack of formation of IgM antibodies and perhaps also to lack of sensitivity of the test. Pyndiah et al. (11) recently reported that, in some sera that are negative in the IgM-IFA test, IgM antibodies to T. gondii can be detected after separation of IgM from other immunoglobulins by gel filtration. Sera in that study were from patients with high titers in the conventional indirect fluorescent antibody test and with clinical illnesses suggestive of toxoplasmosis. We have confirmed and extended the findings of Pyndiah et al. (11) by studying sera that were negative in the IgM-IFA test and obtained from patients with well-defined episodes of acquired toxoplasmosis (10). These sera were obtained at various intervals after onset of symptoms and signs of the infection. We also studied sera that were negative in the IgM-IFA test and obtained from infants with congenital toxoplasmosis. MATERIALS AND METHODS Sera selected for gel filtration. A total of 68 sera from 47 patients with acquired toxoplasmosis were collected during acute illness and at various intervals thereafter. These 68 sera were negative (<1:10 or <1:16) in the IgM-IFA test when first tested in our 336

VOL. 12, 1980 laboratory, although other sera from many of the patients were positive. Of the 47 patients, 26 had lymphadenopathy with histological features characteristic of toxoplasmic lymphadenitis (7) and serological evidence (Welch et al., in press) of acute acquired toxoplasmosis. (All biopsy specimens were reviewed by Ronald Dorfman, Department of Pathology, Stanford University School of Medicine.) The remaining 21 patients had clinical features (10) and diagnostic serological evidence (Welch et al., in press) of acute acquired toxoplasmosis. All 47 patients had Sabin- Feldman dye test (DT) titers of 21:1,000 and IgM-IFA test titers of 21:80 in sera drawn within 6 months of onset of symptoms and signs of toxoplasmosis. When clinical syndromes suggested that rheumatoid factor (4) or antinuclear antibody (2) may have been present, the sera were tested, and those with rheumatoid factor or antinuclear antibody were excluded. Thirteen sera from 13 adults were negative in the DT. These individuals were considered uninfected. Thirteen sera from seven infants, ages 0 to 9 months, with congenital toxoplasmosis were negative (<1:2) in the IgM-IFA test when first tested in our laboratory. DT titers in these sera ranged from 1:64 to 1:16,000. Seven of these sera had been collected by Georges Desmonts (Institut de Puériculture de Paris, France), were found to be negative (<1:2) in the IgM- IFA test when first tested by him, and were stored in his laboratory for 6 to 18 months before they were sent to us. Criteria for the diagnosis of congenital toxoplasmosis were as previously described (12). Seven sera from seven neonates initially suspected of having congenital toxoplasmosis had high titers (1:128 to 1:4,096) in the DT and negative results in the IgM-IFA test and were used as controls. On follow-up, none of these neonates was found to have been infected with T. gondii. Sera selected for treatment with protein A. Twelve sera that were positive in the IgM-IFA test were from 12 patients with acute acquired toxoplasmosis, and five sera were from five infants with congenital toxoplasmosis. Four sera that were negative in the IgM-IFA test had been obtained from four patients 9 to 24 months after onset of acute acquired toxoplasmosis, and 14 were from 14 infants with congenital toxoplasmosis. Criteria for diagnosis of toxoplasmosis were the same as described above. Serological tests. The DT (micromodification; E. Handman and J. S. Remington, Immunology, in press) and the IgM-IFA test were performed within 1 week of receipt of the sera. Sera were then stored at -70 or -20 C for periods of up to 12 years. Certain of the sera had been thawed and frozen more than once. The majority had been tested in the IgM-IFA test using transmission fluorescence microscopy, as previously described (Welch et al., in press). More recent specimens have been tested using Ploém illumination (Zeiss fluorescence microscope with an HBO 200 mercury lamp, BP 450-490 exciter and LP 520 barrier filters, and FT 510 chromatic beam splitter). When examined by transmission fluorescence microscopy, sera were tested in twofold dilutions from 1:10 to 21:160, and a titer of 21:10 was considered suggestive of recent infection. Higher titers are obtained with Ploém illu- DIAGNOSIS OF TOXOPLASMOSIS 337 mination than with transmission illumination. We now routinely use Ploem illumination and fourfold dilutions of serum from 1:16 to 1:4,096. A titer of 1:16 is considered equivocal, and a titer of >1:16 is considered suggestive of recent infection. Sera from infants have always been tested in twofold dilutions, beginning with a titer of 1:2, and a titer of-1:2 is considered diagnostic of congenital toxoplasmosis in the absence of rheumatoid factor or antinuclear antibody (12). Separation of IgM from IgG. A modification of the procedure employed by Pyndiah et al. (11) was used to separate IgM from IgG. Bio-Gel A-5m, 200 to 400 mesh (Bio-Rad Laboratories, Richmond, Calif.) was poured into disposable glass columns with 0.7-cm inner diameters (Econo-column, Bio-Rad) and packed with 3 m of water pressure to a height of 6.5 cm. The gel was equiibrated with buffer containing 0.05 M tris(hydroxymethyl)aminomethane hydrochloride, 0.15 M NaCI, and 0.03% NaN3, ph 7.2. The void volume of the columns (determined with Dextran Blue [Pharmacia Fine Chemicals, Inc., Piscataway, N.J.]) was 0.8 ml. Of each serum sample, 0.1 ml was centrifuged at 10,000 x g (Microfuge; Beckman Instruments, Inc., Fullerton, Calif.) for 5 min, and the supernatant was mixed with 20,ul of Dextran Blue in the buffer (20 mg/ml). The sample was then applied to the column and passed through the gel under 2 m of water pressure and with a flow rate of 150 pl/min. Fractions (50 l) were collected. The columns were washed with at least 10 ml of the buffer after each separation. Preliminary testing revealed that the fraction with the highest concentration of Dextran Blue and the fraction immediately following contained the highest concentrations of IgM [IgM]; these fractions were pooled (IgM preparation) for testing in the IgM-IFA test. Concentrations of IgM antibodies were measured in IgM preparations and in sera by radial immunodiffusion (Endoplates; Kallestad Laboratories, Chaska, Minn.). Detection of IgM-IFA antibodies in IgM preparations. IgM preparations from the columns were tested undiluted and in serial twofold dilutions in the IgM-IFA test with Ploem illumination, as described above. The corresponding sera were tested undiluted and in serial fourfold dilutions in parallel with the IgM preparations. The dilution of IgM antibodies resulting from gel filtration of different sera varied from 2.5- to 10-fold. To correct for this dilution, we estimated the titer of IgM antibodies to T. gondii which, if not blocked, would have been demonstrable in the serum before gel filtration (estimated original titer, EOT) by the following formula: reciprocal of EOT = reciprocal of IgM- IFA titer in IgM preparation x ([IgM] in serum)/ ([IgM] in IgM preparation). The dilutions of IgM in the IgM preparations were always less than 10-fold with respect to the concentrations of IgM in the original sera. Since the EOT is based on this dilution factor, IgM preparations that were negative in the IgM-IFA test are considered as a group to have an EOT of <1:10. Effect of IgG antibodies on detection of IgM antibodies in the IgM-IFA test. To study the effect of IgG antibodies on the IgM-IFA test, preparations

338 FILICE, YEAGER, AND REMINGTON of IgG and IgM, each containing antibodies to T. gondii, were combined before performance of the IgM- IFA test. High-DT titer IgG was extracted by diethylaminoethyl Sephadex chromatography from the serum of a patient with acute toxoplasmosis. The preparation contained 21.5 mg of IgG per ml and had a DT titer of 1:8,096. Low-DT titer IgG was prepared from lyophilized, pooled human 7S globulin (Immunology, Inc., Lombard, Ill.); it contained 21.5 mg of IgG per ml and had a DT titer of 1:256. IgM from serum from another patient with acute toxoplasmosis was separated from IgG by gel filtration; it contained 0.42 mg of IgM per ml and had an IgM-IFA test titer of 1:128. Before performance of the IgM-IFA test, the high-dt titer IgG preparation was serially diluted. An amount of 40,ul of each dilution was mixed with a 40-jil portion of the IgM preparation. Then each mixture of IgG and IgM was serially diluted in fourfold dilutions, and these dilutions were tested in the IgM-IFA test. In the same fashion, 40 gtl of the low-dt titer IgG were mixed with a 40-,ul portion of the IgM preparation, and this mixture was then serially diluted and tested in the IgM-IFA test. Treatment of sera with protein A. Formalinized Staphylococcus aureus (Cowan I), which produces protein A, was used to treat portions of sera diluted 1:10 as previously described (1). The IgM-IFA test was performed on treated and untreated (but similarly diluted) portions of sera in parallel. Concentrations of IgM, IgG, and IgA were measured in treated and untreated portions of radial immunodiffusion as previously described (17). RESULTS Efficiency of gel filtration. Concentrations of IgM in IgM preparations ranged from 11 to 42% of those in the corresponding sera (mean for 54 separations was 18%, representing a mean dilution of 1:6). The gel filtration procedure resulted in separation of IgM from IgG in more than 90% of the specimens. Separation was monitored by testing IgM preparations for IgG by one of the two ways. Certain IgM preparations were tested for IgG by radial immunodiffusion (Quantiplate, Kallestad; lower limit of detection, 0.19 mg/ml). IgM preparations of the remaining sera, all of which contained antibodies to T. gondii, were tested for IgG antibodies to T. gondii by indirect immunofluorescence with goat anti-human IgG as the second reagent (3). When IgM preparations were found to contain measurable IgG, they were not studied further, and portions of the corresponding sera were separated again. Results of a representative separation are shown in Fig. 1. IgM antibodies in IgM preparations of sera from patients with acquired toxoplasmosis. IgM preparations of 68 sera from patients with acquired toxoplasmosis were examined; 36 (53%) were positive in the IgM-IFA test. Titers ranged from positive in the undiluted w 0.2 c, I 0.- [IgG] I oîoîoîoîoîod 2 3 4 5 6 7 8 9 10 FRACTION NUMBER 11-15 FIG. 1. Concentrations of IgM and IgG in selected fractions obtained by gel filtration. (*) denotes the fraction with the highest concentration of Dextran Blue, as determined by visual observation. (O) indicates that IgG was not measurable by radial immunodiffusion. The original serum contained 2.61 mg of IgM and 8.0 mg of IgGper ml. 1-90 80 -à 70 z 0 i 60 o s 50 I n 40 30 2J CC 20 U. w 1 0.4r I0,..@. s- J. CLIN. MICROBIOL. 2 3 4 5 6 7 8 9 10l 1 12 2 3 4 5 6 0 l MONTHS YEARS INTERVAL BETWEEN DATE OF ONSET AND SERUM SAMPLE FIG. 2. Reciprocal EOT for sera that were negative in the IgM-IFA test and obtained at various intervals after onset of symptoms and signs of toxoplasmosis. NEG, IgM preparations negative in the IgM-IFA test when tested undiluted and in dilutions of 1:2 to 1:16. preparations to 1:32. EOT for the 68 IgM preparations ranged from <1:2.5 to 1:192 (Fig. 2). All of the sera from which these IgM preparations were obtained were negative in the IgM-IFA test when first tested. When the sera were retested in parallel with their IgM preparations, 2 (3%) of the sera had titers of 1:4, 8 (12%) were positive undiluted, and the remainder (85%) were nega-

VOL. 12, 1980 tive. There was no correlation between the IgM- IFA test results obtained in these low serum dilutions and the results obtained in their IgM preparations. An EOT of >1:20 was strongly associated with recent onset of acute toxoplasmosis. Of 24 sera obtained from 18 patients within 4 months of onset, 12 (10 patients) had an EOT of >1:20. In contrast, of 44 sera obtained from 37 patients more than 4 months after onset, only 2 (2 patients) had an EOT of >1:20 (P = 0.00003; Fisher's two-tailed exact test). It is noteworthy that of 25 sera obtained from 25 patients more than 1 year after onset, 10 (40%) had IgM antibodies detected in IgM preparations, although 8 of the 10 had an EOT of <1:20. The IgM preparations of the 13 sera that were negative in the DT were tested as controls. IgM preparations of two sera were positive, and the remainder were negative. The EOT for the two sera with positive IgM preparations were low (1:4 and 1:5). IgM antibodies in IgM preparations of sera from infants with congenital toxoplasmosis. IgM preparations of 13 sera from infants with congenital toxoplasmosis were examined; 5 (38%) were positive in the IgM-IFA test. Titers ranged from positive in the undiluted preparations to 1:2. ranged from <1:4.3 to 1:9. All of the sera from which these IgM preparations were obtained were negative in the IgM-IFA test when first tested. When the sera were retested in parallel with their IgM preparations, one of the sera was positive undiluted and the others were negative. When IgM preparations of seven sera from seven uninfected neonates were tested as controls, all were negative in the IgM-IFA test. The mean IgM concentrations in the sera from these neonates were approximately one-fourth of those in the sera from the infants with congenital toxoplasmosis. Effect of IgG antibodies on detection of EOT for the 13 IgM preparations IgM antibodies in the IgM-IFA test. High concentrations of IgG antibodies to T. gondii inhibited detection of IgM antibodies to T. gondii in the IgM-IFA test, and the inhibition was predominantly by specific antibodies to T. gondii rather than by a nonspecific effect of IgG. When portions of purified preparations of high- DT titer IgG containing 21.5 or 2.7 mg of IgG per ml and with DT titers of 1:8,096 and 1:1,024, respectively, were added to the IgM preparation, IgM antibodies to T. gondii were not detected in the IgM-IFA test (Table 1). In contrast, when more dilute portions of this IgG preparation were added to the IgM preparation, IgM antibodies to T. gondii were detected in the IgG- DIAGNOSIS OF TOXOPLASMOSIS 339 IgM mixtures when tested undiluted and in dilutions of 1:4 or 1:16 but not in greater dilutions. To determine whether the inhibition in the IgM- IFA test was due to IgG antibodies to T. gondii or due to a nonspecific effect of IgG, low-dt titer IgG containing 21.5 mg of IgG per ml (the same concentration as in the high-dt titer IgG preparation) but with a DT titer of 1:256 was added to the IgM preparation. IgM antibodies to T. gondii were detected in this IgG-IgM mixture when it was tested undiluted and in dilutions of 1:4 and 1:16 but not in greater dilutions. Since the IgM-IFA test titer was higher (1:64) when the IgM preparation was mixed with phosphate-buffered saline, there may have been some nonspecific binding by IgG or another component(s) of the IgG preparations. However, the differences between the results obtained with the two IgG preparations used at a concentration of 21.5 mg/ml indicate that the major blocking effect was due to IgG antibodies to T. gondii. Effects of treatment with protein A. Treatment with protein A of sera that had been either positive or negative in the IgM-IFA test did not result in an increase in our abiity to detect IgM antibodies in the IgM-IFA test. Treated portions of the sera that were positive in the IgM-IFA test had titers similar to those in the untreated portions. After 18 sera that were negative in the IgM-IFA test had been treated with protein A, IgM-IFA tests were still negative. Concentrations of IgG, IgM, and IgA were lower in treated portions than in untreated portions; the mean decreases (± standard deviation) were 94% (±3%), 39% (±17%), and 22% (±18%), respectively. Thus, the concentrations of IgM antibodies in the treated portions were, on the average, 2.6 times (100/39) less than in untreated portions. Since ail portions were initially diluted by a factor of 10, the concentrations of IgM antibodies in treated portions were actually 26 times less than the concentrations in their respective sera. This dilution of IgM was more than four times greater than the mean dilution (1:6) that resulted from gel filtration. The method of removal of IgG by treatment with protein A was directly compared with the gel filtration method. A serum was obtained 2 months after the onset of symptoms and signs in a patient with toxoplasmic lymphadenitis and was negative in the IgM-IFA test. The IgM preparation (obtained by gel filtration) was positive and the EOT was 1:34. The protein A- absorbed portion of the serum was negative in the IgM-IFA test; the concentration of IgM in this serum was diluted 20-fold by the treatment

340 FILICE, YEAGER, AND REMINGTON J. CLIN. MICROBIOL. TABLE 1. Effect of IgG antibodies to T. gondii on detection of IgM antibodies in the IgM-IFA test Reactivity at DT titer (mg of IgG per ml)": Dilution of IgG-IgM 8,192 (21.5) 1,024 (2.7) 128 (0.34) 16 (0.04) 2 (0.005) 256 (21.5) Controlh Undiluted - - + + + + + 4 - - + + + + + 16 - - + + + + - - 64 + 256 - - - - - 1,024 - - - - - - - Before mixing with IgM preparation. -, Negative; +, positive. h Phosphate-buffered saline, ph 7.2. procedure. From the EOT of 1:34 obtained by gel filtration, one would have expected that the absorbed portion of serum with IgM diluted only 1:20 would have been positive if interfering substances (e.g., IgG) had been completely removed. The amount of IgG that remained (approximately 6%) must have been sufficient to interfere with detection of the remaining IgM antibodies to T. gondii. DISCUSSION Our data confirm that IgM antibodies may not be detected in the IgM-IFA test because of blocking by IgG antibodies to T. gondii. By studying sera obtained at various intervals after onset of symptoms and signs of toxoplasmosis, we have further characterized the diagnostic meaning of IgM antibodies detected only after separation of IgM from IgG. High levels of IgM antibodies to T. gondii in IgM preparations (EOT of >1:20) are strongly suggestive of recently acquired toxoplasmosis. Low levels of IgM antibodies (EOT of s1:20) frequently are present in sera from patients years after onset of toxoplasmosis and in some patients whose sera are negative in the DT. The observation that patients whose sera are negative in the DT occasionally have IgM antibodies which react with T. gondii is not new. These "natural" IgM antibodies, which result in low IgM-IFA test titers, were described previously in whole sera (5, 15). The stimulus or stimuli for these antibodies are not known. Because of the occurrence of natural IgM antibodies, we only consider an IgM-IFA test titer of >1:16 in whole serum to be suggestive of recently acquired toxoplasmosis. We found two patients with a high (>1:20) EOT long after the onset of their symptoms and signs of toxoplasmosis. One had biopsy-proved toxoplasmic lymphadenitis which persisted for 1 year. Six months after onset, he was found to have embryonal cell carcinoma in a testicle. The tumor was resected, extensive evaluation (including pelvic lymphadenectomy) revealed no evidence of spread, and he was not treated further for the tumor. A serum obtained 7 weeks after onset of toxoplasmic lymphadenitis had a DT titer of 1:32,768 and an IgM-IFA test titer of 1:640. A serum obtained 3 years after onset, when the patient was asymptomatic, had a DT titer of 1:4,096 and an IgM-IFA test titer of 1:80. A serum obtained 4 months later had a DT titer of 1:4,096, and the IgM-IFA test was negative. The IgM preparation of the last serum had an EOT of 1:48. The latter serum contained neither rheumatoid factor nor antinuclear antibody. That serum was found to contain T. gondii antigen (2a). Activity of the parasite appeared to persist longer than usual in this patient. The other patient had a mild febrile illness, followed 1 week later by lymphadenopathy. A serum obtained at the time had a DT titer of 1:16,384 and an IgM-IFA test titer of 1:1,280. After 20 months, our laboratory requested a follow-up serum sample. This serum had a DT titer of 1:4,096 and the IgM-IFA test was negative. The IgM preparation of the latter serum had an EOT of 1:35. That serum did not contain demonstrable T. gondii antigen (2a). This patient apparently had an IgM antibody response which persisted longer than usual. When sera positive in the IgM-IFA test were retested after years of storage, we occasionally found lower titers than were found when they were first received. It is likely that the EOT in some sera that are negative in the IgM-IFA test will be higher when separated and tested soon after they are obtained than after storage. This is suggested by the fact that the three highest EOT found in our study were in sera tested within 1 year of receipt in our laboratory, whereas almost all of the other sera had been stored for longer periods of time. Thus, in sera studied soon after they are obtained, the level of EOT which discriminates between patients with recently acquired toxoplasmosis and other patients may be >1:20, the discriminating level found in this study. The detection of IgM antibodies after gel fil-

VOL. 12, 1980 tration in sera that were negative in the IgM- IFA test and obtained from infants with congenital toxoplasmosis indicates that blocking by IgG probably occurs in these sera as well. Detection of antibodies after gel filtration in sera from young infants probably indicates congenital infection. Control sera for these studies were obtained from infants who were younger and had lower serum concentrations of IgM. Although these differences are unlikely to have significantly affected our results, this remains to be clarified. We confirmed the previous observation by Pyndiah et al. (11) that IgG antibodies directly interfere with detection of IgM antibodies to T. gondii in the IgM-IFA test. However, we mixed IgG and IgM before adding the preparations to antigen instead of adding IgG and IgM sequentially because mixing them beforehand more closely approximates adding whole serum to antigen. Moreover, we showed that interference in the IgM-IFA test was predominantly due to IgG antibodies specific for T. gondii rather than due to a nonspecific interference by IgG. Other classes of antibody such as IgA may also interfere, but that possibility was not tested. The methods of gel filtration used in our study and in that by Pyndiah et al. (11) are simple and rapidly performed. Expensive equipment is not required. The disposable columns we used are more expensive than the plastic syringes used by Pyndiah et al. ($3.30 versus $0.15, respectively) but the disposable columns are specifically designed for column chromatography and we found in preliminary tests that they are much easier to use and more efficient than are syringes. Since the columns can be reused several times, their cost should not be a deterrent to their use. Treatment of sera with protein A has been used by numerous investigators as a rapid method for removing IgG. Use of this method did not enable us to detect IgM antibodies to T. gondii more efficiently. Except as specifically noted, the experiments with protein A were performed in different sera than those separated by gel filtration, so results obtained by the two techniques cannot be directly compared. The results suggest that the gel filtration method is more efficient for detecting IgM antibodies to T. gondii in sera that were negative in the IgM- IFA test. This may reflect the fact that, while 94% of IgG was removed from diluted portions of sera, 39% of IgM was removed. This significant removal of IgM has been observed by others (8, 9) and is a detraction of the method whenever relatively low levels of IgM antibodies are present. Our data and those of Pyndiah et DIAGNOSIS OF TOXOPLASMOSIS 341 al. (11) reveal that gel filtration results in far less reduction in the concentration of IgM and greater reduction in the concentrations of IgG and IgA. 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