Table 1.
Several different characteristics of MS patients.
Table 2.
The relative content of different IgGs and anti-MBP antibodies in sera and CSF of patients with MS*.
Figure 1.
Relative content (%) of lambda- (A) and kappa (B) IgGs in the CSFs (▪) and sera (□) of fifteen MS patients.
Total amount of IgGs in the CSFs and sera was taken for 100%. For details, see Materials and methods.
Figure 2.
Relative content (%) of IgG1 and IgG2 (A), IgG3 and IgG4 (B) in sera, as well as IgG1 and IgG2 (C), and IgG3 and IgG4 (D) in CSFs of fifteen MS patients.
Total amount of all types of IgGs in the CSFs and sera was taken for 100%. For details, see Materials and methods.
Figure 3.
Relative content (A450) of anti-MBP IgGs in terms of the same 100-fold dilution for ELISA of CSF and serum preparations of fifteen MS patients.
For details, see Materials and methods.
Figure 4.
SDS-PAGE analysis of homogeneity of csf-IgGmix (7 µg; lanes 2 and 3) and ser-IgGmix (13 µg; lanes 4 and 5) corresponding to 15 CSFs and serum of MS patients in 3–16% gradient gel before (lanes 2 and 4) and after treatment with DTT (lanes 3 and 5) followed by silver staining (A).
The arrows (lane 1) indicate the positions of molecular mass markers. FPLC gel filtration of csf-IgGmix on a Superdex 200 column in an acidic buffer (pH 2.6) destroying immunocomplexes after Abs incubation in the same buffer (B) and csf-IgGmix affinity chromatography on Sepharose bearing mouse IgGs against human IgGs (C): (—), absorbance at 280 nm (A280); (□), relative activity (RA) of IgGs in the hydrolysis of BMP. A complete hydrolysis of MBP was taken for 100%. In-gel assay of MBP-hydrolyzing activity of csf-IgGmix (□; 15 µg) and ser-IgGmix (•; 40 µg) of MS patients and csf-IgGmix (○; 40 µg) (D). The relative MBP-hydrolyzing activity (RA, %) was revealed using the extracts of 2-3-mm fragments of one longitudinal slice of the gel. The RA of IgGs corresponding to complete hydrolysis of MBP was taken for 100%. The second control longitudinal slice of the same gel was stained with Coomassie Blue (lane 1, ser-IgGmix; lane 2, csf-IgGmix). Lane C shows positions of protein markers. The average error in the initial rate determination from three experiments did not exceed 7–10%. For details, see Materials and methods.
Figure 5.
SDS-PAGE analysis of hydrolysis of various proteins by csf-IgGmix (A) and ser-IgGmix (B).
Proteins (0.7 mg/ml) were incubated for 3 h without Abs (odd numbers), 0.03 mg/ml csf-IgGmix (A) or 0.15 mg/ml ser-IgGmix (even numbers): MBP (lanes 1 and 2), hen egg lysozyme (lanes 3 and 4); human milk lactalbumin (lanes 5 and 6), and human milk lactoferrin ((lanes 7 and 8) Lane C corresponds to a mixture of standard protein markers with known molecular masses.
Figure 6.
SDS-PAGE analysis of MBP-hydrolyzing activity of seven individual IgGs from different CSFs of MS patients (A) and healthy donors (B).
The reaction mixtures were incubated with the IgGs 1 and 3 (0.02 mg/ml) or preparations 6, 7, 8, 10, and 11 (0.01 mg/ml) (A) as well as with the csf-IgGmix (lane 1, 0.1 mg/ml) or ser-IgGmix (lane 2, 0.1 mg/ml) of healthy donors (B); lane 2, csf-IgGmix (0.01 mg/ml) of MS patients (B) for 3 h at 35°C. Lanes C (panels A and B) correspond to MBP incubated without Abs. Dependence of the relative MBP-hydrolyzing activity of csf-IgGmix on its concentration (C). The average error in the initial rate determination from three experiments did not exceed 7–10%. For details, see Materials and methods.
Table 3.
The relative specific activities (RAs) and apparent catalytic constants characterizing IgGs from the CSFs and sera of different MS patients in the hydrolysis of MBP*.