Detailed Immunoassay and Western Blot Methods
Preparation of Serum Samples
Blood samples were obtained from the patients by venipuncture
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
Assays for Antibodies to Toxoplasma gondii and
Class specific IgG, IgM, and IgA antibodies to purified T
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
the Mann-Whitney U test.
Western blots were performed for the measurement of IgG and
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,
22kd and 6kd (Figure 1)
1. Jones-Brando LV, Yolken RH.
Laboratory diagnoses of viral infection. IN: Galasso GH, Whitley RJ,
Merigan TC (eds). Antiviral Agents and Human Viral Diseases, Fourth
Edition, Lippencott-Raven Publishers, Philadelphia, Chapter 5, pp. 129-173,
2. Partanen P, Turunen HJ, Paasivuo RT,
Leinikki PO. Immunoblot analysis of Toxoplasma gondii antigens by human
immunoglobulins G, M, and A antibodies at different stages of infection.
J. Clin. Microbiol 1984;20(1):133-135.