Detection of Transaldolase Specific Antibodies in Patients With MS

By Western blot analysis, Abs to affinity-purified rTAL-H were found in 29/94 sera samples from patients with MS, and Abs to MBP were undetectable in sera of these same patients (8). Anti-TAL-H Abs were undetectable in sera samples of appropriate negative controls, including 10 patients with SLE, 9 patients with ONDs, and 74 control healthy blood donors (8). Sera reactivity to rTAL-H of patients with MS was compared to positive control rabbit sera raised against rTAL-H (4,8). TAL-H-specific seropositivity is based on immunoreac-tivity to 500 ng rTAL-H per lane at serum dilutions of 100-fold or higher.

3.1.1. Prokaryotic Expression of rTAL-H

Briefly, full-length TAL-H protein, coding for 337 aa, was expressed as a fusion protein with GST encoded by pGEX-2T plasmid vector (4,11). Optimum stimulation of expression of the recombinant fusion protein was obtained with 1 mM isopropylthio-^-galactoside for 4 h at 37°C.

The TAL-H/GST fusion protein was affinity purified through binding of GST to glutathione-coated agarose beads and cleaved from GST by 1 NIH unit of thrombin (4,12). Functional activity of each batch was analyzed in the transaldolase enzyme assay (7), showing a specific activity of more than 10 U/ mL protein (see Note 1).

3.1.2. Western Blot Analysis of MS Patient Sera vs rTAL-H

1. Load 500 ng rTAL-H protein in 10 ^L SDS sample buffer per well; separate by SDS-PAGE (polyacrylamide gel electrophoresis) and electroblot onto nitrocellulose membrane (8,13).

2. Incubate nitrocellulose strips in T-TBS and 5% skim milk with either patient or donor sera (at a 100-fold or greater dilution) overnight at room temperature (8).

3. Incubate separate strips overnight at room temperature with positive control rabbit anti-TAL-H Ab (4) (see Note 2)

4. Wash nitrocellulose membrane vigorously six times with T-TBS.

5. For detection using rabbit Ab, incubate with HRP-conjugated goat antirabbit IgG at a 1000-fold dilution in T-TBS and 5% skim milk (8).

6. For detection using human Abs, incubate with biotinylated goat antihuman serum and subsequently with HRP-conjugated streptavidin, both at 1000-fold dilutions in T-TBS and 5% skim milk (8).

7. Wash nitrocellulose membrane vigorously six times with T-TBS between incubations.

8. Develop the blots with chloronapthol development solution (see Fig. 1).

9. The presence of transaldolase-specific autoantibodies in patients with MS was compared with patients with ONDs and healthy control donors using a chi-square test.

Fig. 1. Western blot reactivity to affinity-purified 38-kD full-length rTAL-H (A, 500 ng/lane) and human MBP (B, 500 ng/lane) of sera from patients with MS at a dilution of 1:100. + Control indicates rTAL-H-specific rabbit antibody in A and MBP-specific rabbit antibody in B; - Control indicates normal human serum (8).

Fig. 1. Western blot reactivity to affinity-purified 38-kD full-length rTAL-H (A, 500 ng/lane) and human MBP (B, 500 ng/lane) of sera from patients with MS at a dilution of 1:100. + Control indicates rTAL-H-specific rabbit antibody in A and MBP-specific rabbit antibody in B; - Control indicates normal human serum (8).

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