Detailed Immunoassay and Western Blot Methods

a mean of 16+/- 10.2 days following their first admission to the hospital. 

Serum was separated from whole blood shortly after collection and stored at

-80° until testing.  All sera were tested and found negative for

antibodies to HIV-1 and HTLV-1 by immunoassay.  Samples were run under

gondii were measured by enzyme immunoassay using previously described

procedures (1).  The reagents for the toxoplasma assays were obtained from

Viro-Immun Labor-Diagnostika GambH, Oberursel, Germany.  Briefly, purified

antigens from T gondii were immobilized onto the wells of microtiter

plates, wer reacted with a 100ul aliquot of patient serum diluted 1:100 in

phosphate buffered saline containing 0.5% Tween 20 (PBST).  Following

incubation for 30 minutes at 23°, the solid phase surfaces were washed with

PBST and incubated with 100 ul aliquots of peroxidase labeled antibody conjugate

specifically reactive with human IgG, IgM or IgA immunoglobulins. 

Following another incubation for 30 minutes at 23° , the solid phase surfaces

were again washed with PBST and incubated with Trimethylbenzadine-H₂O₂

enzyme substrate.  Following an incubation for 10 minutes at 23°C the

amount of color generated by the ensuing enzyme-substrate reaction was

quantitated by means of a microplate colorimeter at a wavelength of 620nm. 

The results of each assay were expressed in terms of optical density

units.  For categorical determinations, a sample was considered to have an

elevated level of class specific antibody if it generated an optical density

which was greater than the 90th percentile optical density generated by the

sample in the normal control group.  Antibodies of the IgG class were also

measured antigens derived from Herpes Simplex Virus Type 2, Epstein Barr Virus,

Human Herpesvirus Type 6, Rubella Virus, and uninfected RD rhabdomyosaracoma

cells.  These assays were performed in a manner analogous to that of the

assays for T gondii except that these other antigens were immobilized

onto the solid phase in place of the antigens from T gondii. 

Statistical analyses between the case and control populations were performed by

IgM class antibodies to T gondii using previously described methods

(2).  An aqueous extract of T. gondii tachyzoites (Strain RH, Ross Southern

Labs, Salt Lake City, UT) at 10ug/ml in 0.25 M Tris, pH 8.5, 1.1 M glycerol, 73

mM lithium dodecyl sulfate, 0.5 mM EDTA, 0.1% dithiothreitol was resolved by

SDS-PAGE on NuPAGE® 4-12% gradient Bis-Tris gels as described by the

manufacturer (Invitrogen, Carlsbad, CA), proteins were transferred to

nitrocellulose (Schleicher and Schuell, Keene, NH) in 25 mM Tris, pH 8.3, 0.192

M glycine, 20% methanol, and the membranes were blocked with 5% non-fat dry milk

in PBS, and cut into 3 mm strips.  Strips were incubated overnight at room

temperature with constant agitation in 1.0 ml of serum samples diluted 1/100 in

PBS containing  5% milk, 0.% Triton X-100, 0.2% Tween 0, 1.0% BSA, washed 3

times with 1.5 mM imidazole, pH 7.4, 37.8 mM NaCl, 0.025% Tween 20, and bound

antibody was reacted with 1.0 ml of horseradish peroxidase-goat anti-human IgG

(gamma chain specific, 1/4,000) or IgM (mu chain specific, 1/1,000) antibody

(KPL, Gaithersburg, MD) for 1 hr at room temperature.  Strips were washed 3

times, developed with 1.0 ml of TMB membrane substrate (BioFX Laboratories,

Randallstown, MD) and T gondii antigens were identified by co-migration

of prestained protein molecular weight markers and reference to published

results.  Major antigen bands detectable are ones migrating at 45kd, 34kd,