Fig. 2. Recognition by rabbit anti-TAL-H antibody 12484 of 33 cellulose-bound synthetic peptides overlapping TAL-H. The peptides were incubated with Ab 12484 at a 3000-fold dilution. After washing, the strips were incubated with HRP-conjugated goat antirabbit IgG, developed with a chemiluminescent substrate, and exposed to X-ray film. Quantification of antibody reactivities with each peptide was conducted with a computerized automated densitometer (18).
TAL-H and inhibits its catalytic activity (12). In Western blot assay, recognition by the rabbit anti-TAL-H sera of individual TAL-H peptides was assessed at a 3000-fold dilution by chemiluminescent detection (see Fig. 2).
Peptide reactivity with anti-TAL-H Abs was quantified by automated densitometry and expressed as relative binding intensity on a 0-2 scale. Immunodominant epitopes with high binding affinity to anti-TAL-H Abs were defined as having a binding intensity of 1 or higher, that is, 10-fold or more over background.
Subsequently, four immunodominant B-cell epitopes of TAL-H were identified by autoantibodies of patients with MS. Two of these peptides were recognized by both MS sera and rabbit anti-TAL-H sera (18).
3.2.1. Mapping of Immunodominant B-Cell Epitopes in TAL-H and B-Cell Epitopes in Patients With MS
1. Bind the 33 TAL-H overlapping synthetic peptides (32 are 15 aa long, 1 is 17 aa long) to cellulose strips via (p-Ala)2 spacers using the Spot Synthesizer (see Note 3).
2. Wet membranes in methanol for 10 min.
4. Incubate peptide-containing strips with sera of control donors and sera of MS patients having Ab reactivities to human rTAL-H at a 1000-fold dilution or with positive control polyclonal rabbit anti-TAL-H Ab at a 3000-fold dilution in T-TBS and 5% skim milk at room temperature overnight. Additional controls included sera from TAL-H Western blot-negative patients with MS and preimmune rabbit serum.
5. Wash the strips vigorously six times with T-TBS.
6. For detection using rabbit Ab, incubate with HRP-conjugated goat antirabbit IgG.
7. For detection using human Abs, incubate with biotinylated goat (Fab()2 fragments directed against human IgA, IgG, and IgM and subsequently with HRP-conjugated avidin.
8. Between the incubations, vigorously wash the strips six times in T-TBS.
9. Incubate the blots with ECL chemiluminescent substrate.
10. Develop by exposure to X-ray film.
11. Quantify Ab reactivities with each peptide using a computerized automated den-sitometer.
12. Express peptide reactivity with Ab as relative binding intensity on a 0-2 scale (see Note 4).
13. Compare reactivity levels of polyclonal rabbit sera with human sera peptide recognition (see Note 5).
3.2.2. Analysis of Amino Acid Sequence Homologies Between Viral Peptides and Immunodominant B-Cell Epitopes of TAL-H Recognized by Patients With MS
Protein comparison with viral peptides can be investigated using computer programs designed to reveal percentage homologies and position of identical residues per sequence alignment. To analyze the identified immunodominant B-cell epitopes, the GAP program of UWGCG Software was applied (9). All four immunodominant peptides from Subheading 3.2.1. showed sequence homology to viral antigens (see Fig. 3).
3.2.3. Direct Assessment of Molecular Mimicry Between TAL-H B-Cell Epitopes and Related Viral Peptides
To investigate crossreactivity, immunodominant TAL-H epitopes and related viral peptides (see Subheading 3.2.2.) were synthesized onto cellular membranes and tested in parallel for comparative recognition by rabbit anti-TAL-H sera and MS sera (see Subheading 3.2.1.). Correlations of crossreactivities between TAL-H and viral peptides were analyzed with Pearson's multivariate x2 test (18,20).
Resultant data provided direct evidence of MS crossreactivity between common viral antigens and the immunodominant epitope of TAL-H in a subset of patients with MS (18). As a control, sera of patients with MS who lacked TAL-H autoantibodies failed to recognize viral peptides (18).
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